FreeStyle

User Guide
Software Version 1.8

XCALI-98282 Revision A January 2021

© 2021 Thermo Fisher Scientific Inc. All rights reserved.

Foundation, FreeStyle, and mzVault are trademarks, and Orbitrap, Thermo Scientific, TSQ Endura, TSQ
Quantiva, mzCloud, and Xcalibur are registered trademarks of Thermo Fisher Scientific Inc. in the United
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All other trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

Thermo Fisher Scientific Inc. provides this document to its customers with a product purchase to use in the
product operation. This document is copyright protected and any reproduction of the whole or any part of this
document is strictly prohibited, except with the written authorization of Thermo Fisher Scientific Inc.
The contents of this document are subject to change without notice. All technical information in this
document is for reference purposes only. System configurations and specifications in this document supersede
all previous information received by the purchaser.

This document is not part of any sales contract between Thermo Fisher Scientific Inc. and a purchaser. This
document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of
Sale shall govern all conflicting information between the two documents.

Release history: Revision A, January 2021

General Laboratory Equipment. Not intended for use in diagnostic procedures.

 C

Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Accessing Documentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
System Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Special Notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Contacting Us . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Chapter 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Mass Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Base Peak. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Neutral Losses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Effect of Ionization Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Adduct Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Effect of Isotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Isotope Patterns in High-Resolution Data . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Analysis Modes for the Mass Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Full Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Selected Ion Monitoring (SIM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
MS/MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Qualitative Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
New Features and Enhancements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Chapter 2 Using the FreeStyle Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Startup Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Communicator Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Opening Raw Data Files or Sequence Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Factory Default Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Hierarchy of the Chromatogram and Spectrum Views . . . . . . . . . . . . . . . . . . . 32
Using Workspace Pages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Arranging Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Selecting the Columns to Display in a View or Dialog Box with
Tabular Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Saving and Applying Layout Templates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Restoring the Default Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Using the Pointer to Update the Timebase or Rescale a Graph . . . . . . . . . . . . . 41

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Adding Text, Graphic, and Structure Annotations to a Graphical View . . . . . . 41

Copying, Exporting, and Printing Graphical Images and Tabular Data. . . . . . . 46
Copying an Image of a Graphical View to the Clipboard. . . . . . . . . . . . . . . . 47
Copying Tabular Data to the Clipboard . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Exporting or Printing the Contents of a View or Workspace . . . . . . . . . . . . . 47
Setting FreeStyle as the Default Data Visualization Application . . . . . . . . . . . . 49
Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Workspace Options Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Workspace Processing Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Display Options Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Zoom Options Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Text and Graphic Annotation Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Views. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Info Bar Pages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Rearranging the Info Bar Pages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Hiding the Info Bar Pages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Docking or Floating the Info Bar Pages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Chapter 3 Reviewing Chromatographic Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61

Adding Chromatogram Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Closing Chromatogram Views. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Adding Chromatogram Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Adding Chromatogram Traces Manually. . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Adding Chromatogram Traces with the Auto Filter Feature . . . . . . . . . . . . . 65
Deleting Chromatogram Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Defining a Chromatogram Trace from the Chromatogram Ranges View . . . . . 69
Manually Defining Chromatogram Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Defining Chromatogram Ranges by Using a Spreadsheet File . . . . . . . . . . . . 72
Displaying Mass Defect Filtered Chromatogram Traces . . . . . . . . . . . . . . . . . . 73
Applying Mass Defect Filter in a Chromatogram. . . . . . . . . . . . . . . . . . . . . . 75
Linking a Spectrum View to an MDF Chromatogram Trace. . . . . . . . . . . . . 78
Setting up Instrument Status Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Displaying an EIC Trace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Using the Scan Filters Page to Display a Filtered Chromatogram . . . . . . . . . . . 84
Setting Up the Display Options for a Chromatogram Trace . . . . . . . . . . . . . . . 85
Formatting Chromatogram Plots. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Labeling Chromatogram Peaks or Local Maxima. . . . . . . . . . . . . . . . . . . . . . 86
Filling the Chromatogram Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Normalizing Chromatogram Traces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Changing the Y-Axis Title and Scale of the Chromatogram View . . . . . . . . . 95
Changing the Zoom Level of a Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . 96
Automatically Detecting and Integrating Chromatographic Peaks. . . . . . . . . . . 97
Selecting the Manual Noise Region for the Genesis and ICIS Algorithms . . . . 101

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Manually Adding, Undoing, and Deleting Chromatographic Peaks . . . . . . . . 102

Manually Adding Chromatographic Peaks. . . . . . . . . . . . . . . . . . . . . . . . . . 102
Undoing the Last Manually Added Peak . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Deleting Chromatographic Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Importing Components from a Processing Method . . . . . . . . . . . . . . . . . . . . 104
Exporting Scans for a Filtered Chromatogram to a New Raw Data File. . . . . . 105
Working with Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Creating a New Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Comparing Chromatogram Traces in a Sequence . . . . . . . . . . . . . . . . . . . . 107
Chromatogram-specific Toolbars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Chromatogram – Display Options Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . 109
Chromatogram – Workspace Processing Toolbar . . . . . . . . . . . . . . . . . . . . 113
Sequence Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Chromatogram-Specific Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Chromatogram View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Chromatogram Ranges View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Peaks List View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Chromatogram-specific Pages in the Info Bar . . . . . . . . . . . . . . . . . . . . . . . . . 128
Detector Type Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Trace Type Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Scan Filters Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Peak Detection Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Genesis Peak Detection Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
ICIS Peak Detection Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
Avalon Peak Detection Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
PPD Peak Detection Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
Sequence File Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149

Chapter 4 Reviewing Spectral Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151

Displaying the Scan for a Time Point in a Chromatogram . . . . . . . . . . . . . . . 152
Adding Spectrum Views to the Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Selecting Spectra from the Spectrum Ranges Dialog Box. . . . . . . . . . . . . . . . . 153
Creating a MultiSpectrum View and Changing Its Spectrum Plots . . . . . . . . . 154
Formatting a MultiSpectrum View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
Linking and Releasing Spectra to and from a Chromatogram . . . . . . . . . . . . . 157
Linkage States for a Spectrum Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Releasing a Linked Spectrum from the Chromatogram . . . . . . . . . . . . . . . . 158
Linking a Spectrum View to a Chromatogram . . . . . . . . . . . . . . . . . . . . . . 158
Averaging Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Creating an Average Spectrum from the Chromatogram Shortcut Menu. . . 159
Creating an Average Spectrum from the Spectrum Ranges Dialog Box . . . . 160
Subtracting Background Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Using the Tools Commands to Subtract Background Spectra . . . . . . . . . . . 161
Using the Spectrum Ranges Dialog Box to Subtract Background
Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

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Exporting a Selected Spectrum to a New Raw Data File . . . . . . . . . . . . . . . . . 163

Using Precursor Markers to Open Data-Dependent Scans . . . . . . . . . . . . . . . 164
Using the Precursor Flag Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
Using the Nearby Precursors Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
Selecting Spectra from an MSn Tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Specifying the Display Options for a Spectrum View . . . . . . . . . . . . . . . . . . . 175
Modifying the Scan Header for the Spectrum View. . . . . . . . . . . . . . . . . . . 176
Applying Local or Global Normalization in a MultiSpectrum View . . . . . . 177
Labeling Spectrum Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Labeling APD Data in Spectrum Peaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Labeling Mass Spectrum Peaks with Chemical Formulas. . . . . . . . . . . . . . . 180
Changing the Y-Axis Scale of a Spectrum View . . . . . . . . . . . . . . . . . . . . . . 183
Changing the Zoom Level in a Spectrum View or MultiSpectrum View . . . . . 183
Spectra-specific Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Spectrum – Workspace Processing Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . 185
Spectrum – Display Options Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
MultiSpectrum – Display Options Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . 190
Spectra-specific Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
Spectrum View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
MultiSpectrum View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Spectrum Ranges Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
MSn Browser Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

Chapter 5 Reviewing Map Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199

Working with the Map View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Adding Map Views to the Workspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Linking and Releasing Map to and from a Chromatogram . . . . . . . . . . . . . . . 201
Linking Status of a Map View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201
Releasing a Linked Map View from the Chromatogram . . . . . . . . . . . . . . . 202
Linking a Map View to a Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Changing the Zoom level of a Map View . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Map View – Specific Toolbars. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Map View – Workspace Options Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Map View – Display Options Toolbar. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Map-Specific Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Map Ranges Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
Setting Map Ranges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Defining the Retention Time and Mass Ranges. . . . . . . . . . . . . . . . . . . . . . 210
Saving a Map View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Setting the Map Display Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Setting the Format Options. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Setting the Map Axis Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Setting the Map Color Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Setting the Band Width. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215

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Chapter 6 Analyzing MS Data using Data Analytics View . . . . . . . . . . . . . . . . . . . . . . . . . .217

Adding Data Analytics View to the Workspace . . . . . . . . . . . . . . . . . . . . . . . . 217
Plotting the Data as a Histogram. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
Plotting the Data in a Trend Chart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Data Analytics View - Specific Toolbars . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Data Analytics View – Workspace Options Toolbar . . . . . . . . . . . . . . . . . . 219
Data Analytics View – Display Options Toolbar . . . . . . . . . . . . . . . . . . . . . 220
Saving a Data Analytics Plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Data Analytics Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Filtering Data Analytics Views by Trace Types . . . . . . . . . . . . . . . . . . . . . . 222
Creating a Data Analytics Plot. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Chapter 7 Determining the Elemental Composition of Ions. . . . . . . . . . . . . . . . . . . . . . . . . .229

Overview of an Elemental Composition Analysis. . . . . . . . . . . . . . . . . . . . . . . 229
Starting an Elemental Composition Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Using the Fragments Matching Algorithm for Confirmation. . . . . . . . . . . . . . 231
Reviewing the Elemental Composition Results . . . . . . . . . . . . . . . . . . . . . . . . 233
Reviewing the Best-Matching Formulas. . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Intensity Tolerances. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Reviewing the MS/MS Coverage Score for Matching Fragments . . . . . . . . . 238
Reviewing MS/MS Annotation Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Modifying the Elements in the Elements in Use Table . . . . . . . . . . . . . . . . . . 238
Adding Elements to the Elements in Use Table . . . . . . . . . . . . . . . . . . . . . . 239
Removing Elements from the Elements In Use Table . . . . . . . . . . . . . . . . . 240
Using Custom Periodic Table for Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Elemental Composition Results View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Simple Elemental Composition Results View . . . . . . . . . . . . . . . . . . . . . . . . . 245
Elemental Composition Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

Chapter 8 Searching Mass Spectrum Libraries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .253

Performing a Local NIST or mzVault Library Search . . . . . . . . . . . . . . . . . . . 253
Setting Up the Default Library Search Parameters . . . . . . . . . . . . . . . . . . . . 253
Selecting the Query Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Starting a Library Search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Reviewing the Results of a Local NIST or mzVault Library Search . . . . . . . . . 256
Reviewing the Results of a NIST Library Search . . . . . . . . . . . . . . . . . . . . . 256
NIST Search Results View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Reviewing the Results of an mzVault Search . . . . . . . . . . . . . . . . . . . . . . . . 258
mzVault Search Results View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
Modifying the Settings for a Local NIST or mzVault Library Search. . . . . . . . 261
Modifying a NIST Search from the NIST Search Page . . . . . . . . . . . . . . . . 262
Modifying an mzVault Search from the mzVault Search Page . . . . . . . . . . . 266
Searching the Online mzCloud Mass Spectral Database . . . . . . . . . . . . . . . . . 272
Exporting a Mass Spectrum to the NIST MS Search Application . . . . . . . . . . 273
Managing Libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Spectrum Workspace Processing Toolbar – Library Search Features . . . . . . . . 276

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Chapter 9 Simulating Isotope Distributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .277

Simulating the Isotope Distribution for a Chemical Formula or Peptide . . . . . 277
Displaying Centroid and Profile Spectra in the Same Window . . . . . . . . . . . . 283
Isotope Simulation Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

Chapter 10 Working with CV Plots for FAIMS Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .291

CV Traces Dialog Box. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
CV Plot View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Export CV Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Chapter 11 Peptide Fragment Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .297

Overview of Peptide Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Fragmentation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Annotating Peptide Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Peptide Fragments Info Bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Peptide Results View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Filtering the Fragmented Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Chapter 12 Viewing Experiment and Instrument Information . . . . . . . . . . . . . . . . . . . . . . . . .305

Workspace Options Toolbar – Report Area. . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Spectrum List – Display Options Toolbar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Scan Header View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Spectrum List View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Status Log View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Instrument Method View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Tune Method View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Sample Information View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
File Header View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Editing the File Header Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
Error Log View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Chapter 13 Running the Xtract Algorithm on Spectra and Chromatogram Data . . . . . . . . .321
Understanding the Xtract Algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Applying the Xtract Algorithm to an Isotopically Resolved Spectrum . . . . . . . 322
Xtract Page for a Selected Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Deconvolved Spectrum View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Xtract Results View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Applying the Xtract Algorithm to a Chromatogram . . . . . . . . . . . . . . . . . . . . 335
Xtract Page for a Selected Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Appendix A FreeStyle Default Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .341

Modifying, Saving, and Resetting the Default Configuration Options . . . . . . 341
Default Mass Precision Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Default Peak Detection Algorithm Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Default Number of Recently Used Files Page . . . . . . . . . . . . . . . . . . . . . . . . . 345

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Default Raw Data Files Directory Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Default Elemental Composition Search Parameter Page . . . . . . . . . . . . . . . . . 347
Factory Default Template Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Default Library Search Parameters Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
Workspace Options Page. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Xtract Options Page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Appendix B Scan Filters and Scan Headers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .359

Scan Filter Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
Scan Headers and Scan Header Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . 362

Appendix C One- and Three-Letter Abbreviations for Amino Acid Residues . . . . . . . . . . . .365

Appendix D Common Isotopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .369

Thermo Scientific FreeStyle User Guide 9

Contents

10 FreeStyle User Guide Thermo Scientific

 P

Preface
This guide describes how to use the Thermo FreeStyle™ application to view and analyze raw
mass spectrometry data.

For contact information and information about related documentation and system
requirements, see these topics.

Contents
• Accessing Documentation
• System Requirements
• Special Notices
• Contacting Us

For a list of new features, see New Features and Enhancements.

Y To open the FreeStyle application

• From the Microsoft™ Windows™ taskbar, choose Start > Thermo FreeStyle >
FreeStyle.
–or–
• Click the FreeStyle icon, , on the desktop.

Accessing Documentation
The FreeStyle application includes complete documentation. In addition to this guide, you
can also access the application Help.

Y To view the product manual

From the taskbar, choose Start > Thermo FreeStyle > FreeStyle User Guide.

Y To open the Help system from the FreeStyle window

Choose File > Help.

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Preface

Y To view context-sensitive Help

If information is available for a specific view, page, dialog box, or toolbar, click Help or
press the F1 key.

Y To view manuals or animations from the website

1. Go to thermofisher.com.
2. Point to Support.
3. On the left, click Manuals.
4. In the Search box, search by the product name.
5. From the results list, click the title to open the document or animation in your web
browser.
To return to the document list, click the browser Back button.

System Requirements
Your system must meet the following minimum system requirements.

System Requirements
Computer • 3.6 GHz quad core processor with a minimum of 8 GB RAM
(16 GB recommended)
• CD/R-ROM or DVD drive
• 1 TB hard drive
• Video card and monitor capable of 1920 × 1080 resolution
Software • Adobe Reader™ 10.1 or later
• Microsoft .NET Framework 4.7.2
• Microsoft Office 2013 (for exported data)
• Microsoft Windows 7 SP1 (64-bit), Windows 10 (64-bit)
• Thermo Xcalibur™ 4.2 SP1 or later (for the NIST™ Library
Browser)
• Thermo Foundation™ 3.1 SP6 or later

12 FreeStyle User Guide Thermo Scientific

 Preface

Special Notices
Make sure that you follow the special notices presented in this guide. Special notices appear in
boxes; those concerning safety or possible system damage also have corresponding caution
symbols.

This guide uses the following types of special notices.

IMPORTANT Highlights information necessary to prevent damage to software, loss of

data, or invalid test results; or might contain information that is critical for optimal
performance of the system.

Note Highlights information of general interest.

Tip Highlights helpful information that can make a task easier.

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Preface

Contacting Us
Contact Email Telephone QR Code

U.S. Technical Support [email protected] (U.S.) 1 (800) 532-4752

U.S. Customer Service [email protected] (U.S.) 1 (800) 532-4752

and Sales

Global support Y To find global contact information or customize your request

1. Go to thermofisher.com.
2. Click Contact Us, select the country, and then select the type of support
you need.
3. At the prompt, type the product name.
4. Use the phone number or complete the online form.

Y To find product support, knowledge bases, and resources

Go to thermofisher.com/us/en/home/technical-resources.

Y To find product information

Go to thermofisher.com/us/en/home/brands/thermo-scientific.
Note To provide feedback for this document, send an email message to Technical Publications
([email protected]).

14 FreeStyle User Guide Thermo Scientific

 1

Introduction
Use the FreeStyle application to visualize and qualitatively analyze mass spectrometry data. A
qualitative analysis focuses on identifying unknown compounds and confirming the presence
of target (expected or known) compounds.

With the FreeStyle application, you can display chromatograms and spectra, detect and
integrate chromatographic peaks, search mass spectral libraries, simulate mass spectra, subtract
background spectra, apply scan filters, annotate plots with text and graphics, create and save
layouts, view the status of various instrument parameters during data acquisition, and create a
2D or 3D representation of an analysis displaying the acquired mass/wavelength scans. You
can also export spectral data to the NIST application or mzCloud.org, calculate the elemental
composition of a component from its exact mass, set the parameters for defining the charge
state for peptide fragment matching, and perform Xtract deconvolution.

For a general understanding of mass spectrometry data and to get started with the application,
see these topics.

Contents
• Mass Spectra
• Analysis Modes for the Mass Spectrometer
• Qualitative Analysis Tools
• New Features and Enhancements

Mass Spectra
There are many different types of mass spectrometry (MS) detectors, but the basic principles
are the same in all cases: the MS ionizes the sample, separates the ions according to their
mass1, and moves the separated ions toward a detector where they are counted. The data
system compiles a spectrum showing the mass distribution of the ions produced from the
sample—a snapshot of ion intensities plotted against their mass-to-charge (m/z) ratios.

1 In the majority of cases z=1 and the x axis becomes equivalent to mass, m.

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1 Introduction
Mass Spectra

Ionization initially produces molecular ions, but complex secondary processes can cause the
molecular ions to fragment. Together with molecular ions, these fragment ions make up the
mass spectrum. For individual chemical substances, a mass spectrum can be a characteristic
molecular fingerprint.

Mass spectra have these common features:

• Base Peak
• Neutral Losses
• Effect of Ionization Modes
• Adduct Formation
• Effect of Isotopes
• Isotope Patterns in High-Resolution Data

Base Peak
To plot the MS detector’s response, the most abundant ion, called the base peak, is given an
arbitrary abundance or intensity of 100. The application reports other peaks as a percentage of
the size of the base peak. After this normalization, the data system can compare spectra
directly.

Figure 1 is an example of a NIST library spectrum showing the fragmentation of acetone

C3H6O (molecular weight = 58 Da). The mass-to-charge labels appear above the spectrum
peaks for the most abundant ions. In this example, the molecular ion (58 Da) is not the most
abundant ion. The most abundant ion is the acetyl ion CH3CO (molecular weight = 43 Da).
Figure 1. 70 eV electron ionization (EI) mass spectrum of acetone

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 1 Introduction
Mass Spectra

Neutral Losses
You can use fragmentation patterns, similar to the pattern displayed in Figure 1 for acetone, to
determine the molecular structure of a compound. For example, the neutral loss of 15 Da
from the molecular ion of acetone indicates the presence of a methyl group in the original
molecule. A subsequent loss of 28 Da corresponds to the loss of CO. Table 1 lists commonly
observed neutral losses, measured by the molecular weight of the compound. Assign such
losses to help deduce the structure of an unknown compound. A full structural analysis
generally relies on the presence of a molecular ion and the measurement of the molecular
weight of the compound.
Table 1. Common neutral losses
Loss Fragment
15 CH3
18 H2O
19 F
28 CO
29 C2H5 or CHO
35 Cl
46 NO2
59 C3H7O, COOCH3 or CH2COOH
77 C6H5

In some cases, fragmentation is extensive, leaving little or no trace of a molecular ion. With no
molecular ion, determining either the molecular weight or the structure is difficult.

Effect of Ionization Modes

The ionization mode affects the spectrum characteristics of a compound. The ionization
modes for LC/MS (liquid chromatograph/mass spectrometer) instruments are different from
those used with GC/MS (gas chromatograph/mass spectrometer) instruments:
• Ionization Modes for LC/MS Instruments
• Ionization Modes for GC/MS Instruments

Ionization Modes for LC/MS Instruments

LC/MS instruments use a variety of ionization techniques, collectively called atmospheric
pressure ionization (API). Detectors of this type can detect positive or negative ions.

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1 Introduction
Mass Spectra

API techniques offer soft ionization, usually with little or no fragmentation. An API spectrum
typically contains peaks for only the protonated or deprotonated molecule. Compounds with
basic sites (such as amines) can form protonated molecules [M+H]+. In the positive ion
detection mode (polarity +), these ions produce a spectrum peak at the m/z value M+1 (where
M represents the molecular weight of the neutral compound).

Compounds with acidic sites (sulphonic acids, for example) can form deprotonated molecules
[M–H]–. In the negative ion detection mode (polarity –), these ions produce a spectrum peak
at the m/z value M–1.

Ionization Modes for GC/MS Instruments

GC/MS instruments offer two techniques: electron ionization (EI) and chemical ionization
(CI).

EI is commonly used because it is simple and reproducible. The fragmentation pattern is

effectively determined by the energy of the impacting electrons alone (electron energy,
measured in eV). Different types of mass spectrometers that use EI can produce virtually
identical spectra as long as they have same electron energy.

This reproducibility has led to an extensive library compilation for 70 eV EI spectra. With the
FreeStyle application, you can access the NIST/EPA/NIH Mass Spectral Library with over
108 000 reference EI spectra. You can use library data to select confirmatory ions for your
target compounds.

Note You can purchase the NIST Mass Spectral Search application from the National
Institute of Science and Technology. Thermo Fisher Scientific provides local versions of
the NIST application and its libraries with the Xcalibur data system.

Chemical ionization (CI) offers a softer method of forming ions. In CI, a controlled flow of a
reagent gas, commonly ammonia, methane, or isobutane, is introduced into the area where
ionization occurs (the ion source). Energetic electrons that pass through the source ionize the
reagent gas, as in EI. These ions can then collide with neutral molecules, causing hydrogen
transfer. This process is repeated when the reagent gas ions collide with analyte molecules.

CI usually produces protonated molecules, generally at a mass one unit greater than the
molecular mass of the compound. Significantly less fragmentation occurs than in comparable
EI spectra. Depending on your choice of reagent gas, adduct ions can form. For example,
when you use ammonia as the reagent gas, M+NH4 is a typical adduct ion.

Under certain conditions, CI produces negative molecular ions formed by electron capture.
The sensitivity of negative ion CI for certain classes of compounds (those containing double
bonds, sulfur, phosphorus, chlorine, or bromine) can be orders of magnitude greater than
positive CI or EI modes for those compounds.

For more information about the ionization modes available on your instrument, read the
hardware manual and the instrument manual on how to get started.

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 1 Introduction
Mass Spectra

Adduct Formation
If ionization takes place in the presence of contaminants or additives, such as ammonium or
sodium ions, some compounds are susceptible to adduct formation. These spectra show other
ions in addition to, or instead of, the molecular ion (Figure 2).

Note The FreeStyle application can automatically add elemental composition and m/z
annotations to the mass spectrum peaks (see Labeling Spectrum Peaks). To add custom
annotations, such as those shown in Figure 2, you can use the application’s text annotation
tools.
Figure 2. Mass spectrum showing sodium and acetonitrile adducts

Table 2 lists common adducts for the positive and negative ESI modes.
Table 2. Common adduct ions
Cationized adducts (positive mode) Anionized adducts (negative mode)
+
[M+NH4] M+18 [M+OAc]– M+18
[M+Na]+ M+23 [M+Na]– M+21
[M+CH3OH+H]+ M+33 [M+Cl]– M+35
[M+K]+ M+39 [M+K]– M+37
[M+CH3 CN+H]+ M+42 [M+HCOO]– M+59

Take care when determining molecular weights to account for possible adduct ions.

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1 Introduction
Analysis Modes for the Mass Spectrometer

Effect of Isotopes
In some cases, the effect of less abundant isotopes might cause you to use an average molecular
weight rather than one based on the most abundant isotopes. When the molecular structure
of the target compound contains large numbers of certain elements, the less abundant
isotopes become significant. This situation might result in a shift in the mass peaks from their
expected m/z values.

For example, the most abundant isotope of chlorine is Cl35. However, Cl37 occurs with a
natural abundance of 24.47 percent. When a compound contains four chlorine atoms, its
molecular ion is two mass units greater than that expected from a calculation based solely on
Cl35. Using chlorine’s average atomic weight (35.453), you can correctly identify the
molecular ion. Also, you observe a distribution of molecular ions across eight mass units from
molecules containing between zero and four Cl37 atoms.

Isotope Patterns in High-Resolution Data

The mass spectra acquired with a high-resolution, accurate-mass (HRAM) mass spectrometer
include isotope clusters for analytes with elements that have more than one stable isotope.
The mass difference between the isotopic peaks is proportional to the mass difference of the
isotopes, and the relative intensity of the isotopic peaks is proportional to the natural
abundance of the isotopes.

To confirm the identity of an unknown analyte, compare the theoretical isotope pattern for its
proposed chemical formula to the experimental mass spectrum. Use the Isotope Simulation
Page in the Info Bar to predict the isotope pattern for any chemical formula or peptide
sequence.

Analysis Modes for the Mass Spectrometer

A Thermo Scientific mass spectrometer has these analysis modes:
• Full Scan
• Selected Ion Monitoring (SIM)
• MS/MS

Full Scan
In full-scan operation, the MS detector scans repetitively over a wide mass range throughout
the analysis and sends the data to the data system computer.

With the FreeStyle application, you can display the chromatograms (measured intensity versus
analysis time) for full-scan MS data in these ways (plot types):

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 1 Introduction
Analysis Modes for the Mass Spectrometer

• As a total ion current (TIC) chromatogram. A TIC chromatogram represents the

summed intensities of all the ions in the scanned mass range (mass spectrum) plotted
against the chromatographic retention time. Each peak in the TIC represents one or more
eluting compounds, which can be identified from the mass spectra recorded across the
peak.
• As a mass chromatogram for a range of masses within the scan range. Mass
chromatograms show the ion intensities of selected mass-to-charge ratios (m/z). The
application extracts these mass spectra from each stored scan and plots them against the
analysis time. Use this technique to increase selectivity by displaying an m/z value that is
characteristic of the compound of interest but not present in other sample components.
• As a base peak chromatogram. Base peak chromatograms show the ion intensities of the
most intense ions for each time point in the chromatogram.

Note The FreeStyle application uses the accurate mass and isotope pattern information in
the full-scan MS1 data to calculate the elemental composition of unknown compounds. It
then uses the accurate mass data for the fragment ions in the data-dependent MS2 scans
to confirm the best matching formulas.

Selected Ion Monitoring (SIM)

In the selected ion monitoring (SIM) mode, the MS detector monitors a limited number of
m/z values that are characteristic of a targeted compound or compounds. During an analytical
run, the mass analyzer repeatedly switches between the selected m/z values and monitors each
m/z value for a programmed dwell time before averaging the measured ion intensities and
moving on to the next value.

SIM generates mass chromatograms of only the monitored m/z values, not complete mass
spectra as in the full-scan mode. Without a complete mass spectrum, you cannot perform a
library search to identify an unknown.

SIM is ideally suited to trace analysis and offers reduced file sizes compared to full-scan
operation because SIM records only the information of interest.

MS/MS
Depending on your instrument, you might also be able to do additional stages of mass
analysis called MS/MS.

In an MS/MS experiment, you select specific ions for further fragmentation while discarding
all other masses. The selected ion is called a precursor (parent) ion and its fragment ions are
called product ions. An ion trap mass spectrometer (a mass spectrometer with an ion trap
mass analyzer) can perform additional stages of MS (called MSn), up to MS10.

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1 Introduction
Qualitative Analysis Tools

The MS detector can monitor the product ions in either the full-scan mode or the SIM mode.
When you set up the MS detector to monitor a specific product ion of a specific precursor
(parent) ion, the scan type is called selective reaction monitoring (SRM).

You can create your own libraries of full-scan MS/MS data to use for matching.

You can display the chromatograms for full-scan MS/MS data in these plot types: TIC, mass
range, base peak, neutral fragment, or mass defect filter. With the neutral fragment plot type,
you must specify the neutral fragment.

Note The query spectrum for a library search against your local mzVault™ database file or
the online mzCloud™ mass spectral library must be an MS2 spectrum from a
data-dependent full-scan MS/MS experiment.

Qualitative Analysis Tools

The FreeStyle application includes the following qualitative analysis tools:
• Automated library searches of local mzVault and NIST mass spectral databases and the
online mzCloud database. See Chapter 8, “Searching Mass Spectrum Libraries.”
• Automated peak detection algorithms for the evaluation of chromatographic data (see
Automatically Detecting and Integrating Chromatographic Peaks).
• Elemental composition tool that determines the chemical formulas of ions by using their
exact mass and isotope pattern. Includes confirmation of the best match formula by
comparing the m/z values of the ions in the data-dependent fragmentation scans of the
precursor ion to the possible set of fragment ions based on their m/z values. See
Chapter 7, “Determining the Elemental Composition of Ions.”
• Isotope simulation tool that displays a simulated mass spectrum from the following input:
chemical formula, adduct species, and charge distribution. You can compare the
simulated spectrum to an experimental spectrum. See Chapter 9, “Simulating Isotope
Distributions.”
• Xtract tool for the deisotoping and deconvolution of mass spectra. See Chapter 13,
“Running the Xtract Algorithm on Spectra and Chromatogram Data.”
• Spectrum averaging for noise reduction and mass accuracy (see Averaging Spectra).
• Background subtraction for the removal of spectral peaks—for example, matrix-related
peaks—that are not related to the target components (see Subtracting Background
Spectra).

22 FreeStyle User Guide Thermo Scientific

 1 Introduction
New Features and Enhancements

New Features and Enhancements

The following features are new in the FreeStyle 1.8 application:
• Display peak labels produced by the APD algorithm that runs on the embedded
instrument computer during data acquisition
(Thermo Scientific™ Orbitrap Exploris™ MS Series: ICSW 4.0 or later and
Thermo Scientific Orbitrap Tribrid™ MS Series: Tune 3.5 or later)
• Elemental composition calculation based on mass only
• Thermo Scientific FAIMS Pro™ interface CV optimization data processing and export for
TSQ ICSW version 3.4 or later
• Apply Xtract All deconvolution to chromatograms
• Peak area label with full numerical display
• MSn browser mass tolerance and precursor masses support 4 decimals
• Improved usability by reorganizing the ribbon menu and right-click menu options
• Backward compatibility with Thermo Scientific Foundation™ software 3.1 SP6 or later

The following features were new in the FreeStyle 1.7 application:

• Data Analytics feature that displays options to analyze the MS trending information
from mass spectrometers. The analysis is available as a Histogram or Trend plot in a
separate window.
• A Mass Defect Filter (MDF) feature is introduced to create a mass defect filtered
chromatogram for one or more user-defined mass and mass defect ranges.
• Modifies the Elemental Composition Info page with the addition of a custom
periodic table that lets you add custom isotope abundance to the elements for
elemental composition calculation and isotope simulations.
• Includes an option to compare the theoretical isotope pattern for the proposed
chemical formula by displaying the matched and missed isotopes in the Spectrum
view.
• A new customizable File Header view that displays information from the acquisition
sequence, the autosampler, and the mass spectrometer.
In addition, these enhancements let you do the following:
• Includes an option to save text and structure annotations in the Spectrum View.
• Includes Ranges as a right-click option in the Map View.
• Modifies the Map Ranges dialog box with the addition of Mass range and
Retention Time value fields.
• Includes a new peak identification group section in the Peak Detection Info bar
page to identify the nearest, highest, or all peaks.
• Includes an option to import component parameters from the processing
method to the current or copy of the selected trace

Thermo Scientific FreeStyle User Guide 23

1 Introduction
New Features and Enhancements

24 FreeStyle User Guide Thermo Scientific

 2

Using the FreeStyle Window

The FreeStyle window includes multiple toolbars, views, and Info Bar pages. These topics
describe the FreeStyle window, the default workspace layout, the hierarchy of the interactive
views, and some common tasks.

Contents
• Startup Window
• File Menu
• Communicator Bar
• Opening Raw Data Files or Sequence Files
• Factory Default Layout
• Hierarchy of the Chromatogram and Spectrum Views
• Using Workspace Pages
• Arranging Views
• Selecting the Columns to Display in a View or Dialog Box with Tabular Data
• Saving and Applying Layout Templates
• Restoring the Default Settings
• Using the Pointer to Update the Timebase or Rescale a Graph
• Adding Text, Graphic, and Structure Annotations to a Graphical View
• Setting FreeStyle as the Default Data Visualization Application
• Toolbars
• Views
• Info Bar Pages

Thermo Scientific FreeStyle User Guide 25

2 Using the FreeStyle Window
Startup Window

Startup Window
The FreeStyle window opens to the Isotope Simulation and Elemental Composition pages in
the Info Bar to the left and the getting started links to the right (Figure 3).

You can use the Isotope Simulation page without opening a raw data file (see Isotope
Simulation Page).

You can use the Elemental Composition page without opening a raw data file (see Elemental
Composition Page).
Figure 3. Startup window

Note This user guide uses the following terms to describe the user interface:
• View—A defined area in a workspace, such a Spectrum view or a Chromatogram
view.
• Page—A tabbed document, for example, a page in the Info Bar or a Workspace page
in the main window. In any of these tabbed groups, only one of these pages is the
active page.
• Dialog box—A graphical element that accepts user input. Only one dialog box can be
open at a time. When it is open, a dialog box blocks you from working in other parts
of the application.

26 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
File Menu

File Menu
Use the File menu (see Figure 4) of the FreeStyle window to open raw data files or sequence
files, create a sequence list, save the active workspace as a template, view the list of recently
opened raw data files and folders, and access the default settings.

For more information about workspaces, see Using Workspace Pages. For more information
about templates, see Saving and Applying Layout Templates. For more information about
default settings, see Appendix A, “FreeStyle Default Settings.”
Figure 4. File menu

Table 3 describes the File menu commands. For information about using each command, see
the related topics.
Table 3. File menu commands (Sheet 1 of 2)
Item Description
Commands
New Workspace Opens a dialog box where you can choose existing raw data files
(.raw) or sequence files (.sld) to open and create new Workspace
page. See Using Workspace Pages.
Create Sequence Opens a dialog box where you can choose a set of raw data files to
open and create a sequence in a new Workspace page. See Working
with Sequences.
Save as Default Automatically saves the current layout of the active Workspace page
to the default template file. See Saving and Applying Layout
Templates.

Thermo Scientific FreeStyle User Guide 27

2 Using the FreeStyle Window
File Menu

Table 3. File menu commands (Sheet 2 of 2)

Item Description
Save As Opens a dialog box where you can save the current layout to a
template file with a different name. In the File Name box, type the
new name and click Save. See Saving and Applying Layout
Templates.
Default Options Opens the Default Options Configuration dialog box, where you
specify the default settings for the application (see FreeStyle Default
Settings).
Print Prints a report showing chromatogram and spectrum information for
the currently selected Workspace page.
Help Opens the FreeStyle Help window.
FreeStyle User Opens the FreeStyle User Guide as a PDF file.
Guide
Report an Issue Opens a mail dialog box in the Microsoft Outlook™ application with
a default email address. You can enter a detailed description of the
issue, along with the steps to reproduce it, and send this information
to Thermo Fisher Scientific.
About FreeStyle Displays the FreeStyle version information and the release and
copyright dates, and shows the version information of other Thermo
Scientific applications and instruments installed on your system.
Close Closes the File menu.
Button
Exit Closes the FreeStyle application.

28 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Communicator Bar

Communicator Bar
The communicator bar (located immediately below the toolbar) provides general information
about the current task or warning messages. This symbol, , precedes general information,
and this symbol, , precedes warnings.

The communicator is displayed by default and is specific to each workspace. When you close
the communicator bar in a workspace, it is not automatically closed in other opened
workspaces.
Figure 5. Communicator bar with informational message

Communicator bar with information

about the current data file

Opening Raw Data Files or Sequence Files

In the FreeStyle application, you can open two file types—raw data files (.raw) or sequence
files (.sld). A raw data file contains the data from a single acquisition run; a sequence file
contains a list of associated raw data files from multiple runs. When you open either of these
file types, the application creates a new Workspace page.

Follow these procedures:

• To open a raw data file or a sequence file
• To open a raw data file to a custom layout
• To open a file from the startup window

Thermo Scientific FreeStyle User Guide 29

2 Using the FreeStyle Window
Opening Raw Data Files or Sequence Files

Y To open a raw data file or a sequence file

1. Do one of the following:

• In the menu bar, choose File > New Workspace.
–or–
• In the Create area of the Workspace Options toolbar, click New Workspace.

New Workspace icon

2. In the Open Raw File dialog box, browse to and select a raw data file (.raw) or a sequence
file (.sld), and then click Open.
• For a raw data file, a new Workspace page appears to the right of the Info Bar and has
two stacked views. When the file includes mass spectrometry data, the
Chromatogram view at the top displays the TIC trace, and the Spectrum view at the
bottom displays the first scan.
• For a sequence file, the Sequence File page appears in the Info Bar with a list of raw
data files, and the Workspace page displays data from the first raw data file in the list.
For information about viewing a trace other than the TIC trace, see Defining a
Chromatogram Trace from the Chromatogram Ranges View.

Y To open a raw data file to a custom layout

1. In the startup window, under New Workspace, click From Layout Template.

Tip The From Layout Template command is available only in the startup window. To
apply a custom layout to an active workspace, from the Workspace Options toolbar,
choose Apply > Named Layout Template.

2. In the Browse Templates dialog box, select a template (XML) and click Open.
3. In the Open Raw File dialog box, select a raw data file and click Open.

Y To open a file from the startup window

Do the following:
• To open a recent file, under Open a Recent Item, click the link to the file.
• To open a raw data file or a sequence file, under New Workspace, click From File.
• To open only a sequence file, under New Workspace, click From Sequence.

30 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Factory Default Layout

Factory Default Layout

Figure 6 shows the default FreeStyle window with two raw data files. Each raw data file
appears as a separate tabbed Workspace page, with only one tabbed Workspace page displayed
at a time.

The default layout displays the tabbed Isotope Simulation, Peak Detection, MSn Browser, and
Detector Type pages in the Info Bar to the left and the stacked Chromatogram and Spectrum
views in the workspace area to the right.

In the title bar for a Chromatogram view or a Spectrum view, the tilde symbol (~) to the left
the view’s name indicates that actions in an interactive view or the toolbar affect this view.

Note To open a new Workspace page, see the previous topic “Opening Raw Data Files or
Sequence Files.”

Figure 6. FreeStyle default window

File menu Communicator bar Workspace page Toolbar tabs Toolbar Chromatogram view

Info Bar Spectrum view

Workspace page

Thermo Scientific FreeStyle User Guide 31

2 Using the FreeStyle Window
Hierarchy of the Chromatogram and Spectrum Views

Hierarchy of the Chromatogram and Spectrum Views

A Workspace page can contain multiple Chromatogram, Spectrum, and MultiSpectrum
views, one each of the report views, and one Chromatogram Ranges view. You can select only
one view on the Workspace page at a time. Selecting a view turns its title bar a darker shade
and adds a tilde to the left of the view’s name.

There are three hierarchical states for the Chromatogram, Spectrum, and MultiSpectrum
views: selected, active, and inactive (Table 4).
Table 4. Hierarchical states for the Chromatogram, Spectrum, and MultiSpectrum views
State Indicated by Description
Selected (and Darker title bar with a tilde (~) to Determines the view-specific toolbars and the view-specific
active) the left of the view’s name and a tools in the Workspace Processing toolbar.
green bar with the selected view’s
name—Chromatogram, Spectrum, The actions in the view-specific toolbars and the tools in the
or MultiSpectrum—above the Workspace Processing toolbar affect the selected view.
toolbar
Active Lightly shaded title bar with a tilde Actions in the selected interactive view affect the active view:
(~) to the left of the view’s name.
• The actions performed in a linked and selected Spectrum
The last selected view of each view
or MultiSpectrum view or the Chromatogram Ranges
type is active.
view affect the active chromatogram plot.
• The actions performed in the selected chromatogram plot
affect the active and linked spectrum.

Choosing Chromatogram > Insert Trace in the Workspace

Options toolbar adds a chromatogram trace to the active or
selected Chromatogram view.

Choosing Spectrum > Multi Spectrum in the Workspace

Options toolbar changes the active or selected Spectrum view
to a MultiSpectrum view.

Choosing Chromatogram > Insert View or Spectrum > Insert

View in the Workspace Options toolbar adds a copy of the
active Chromatogram view or the active Spectrum view,
respectively, to the current Workspace page.
Inactive Lightly shaded title bar without a Unaffected by the toolbar actions or actions in other views.
tilde

32 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Using Workspace Pages

Using Workspace Pages

The FreeStyle application creates a new Workspace page whenever you open a raw data file or
a sequence of raw data files. The Workspace page displays the processed data for the associated
raw data files (see Factory Default Layout).

Follow these procedures:

• To create multiple workspaces
• To reposition the Workspace page
• To display a Workspace page when multiple workspaces are open
• To refresh the Chromatogram or Map View for a workspace
• To close a Workspace page

Y To create multiple workspaces

1. Choose File > New Workspace.

2. Do one of the following:
• Select multiple raw data files and click Open.

Tip Use the SHIFT key (for consecutive files) or the CTRL key (for
nonconsecutive files).

–or–
• Drag the files into the FreeStyle window.

Note When you drag files into the Chromatogram Ranges view, this action adds
new traces to the view instead of adding new Workspace pages.

A separate Workspace page appears for each raw data file. In the default factory layout,
additional workspaces appear as horizontal tabs above the Chromatogram view.

Y To reposition the Workspace page

1. Right-click the Workspace page title bar or click its Window Position icon, , to open
the shortcut menu.
Figure 7. Workspace page shortcut menu
Workspace page title bar

Shortcut menu

Thermo Scientific FreeStyle User Guide 33

2 Using the FreeStyle Window
Using Workspace Pages

2. Do one of the following:

• Choose Floating to detach the Workspace page into a floating window.
• Choose Dockable to return the Workspace page back to its default position in the
FreeStyle window.
–or–
• Choose Auto Hide or click the Auto Hide (vertical pin) icon, , in the title bar
(Figure 8) to temporarily hide the Workspace page.
Figure 8. Workspace vertical Auto Hide icon
Auto Hide icon

The Workspace page collapses as a vertical tab to the right of the Info Bar (Figure 9).
Figure 9. Workspace tab
Info Bar

Vertical Workspace page

tab when Auto Hide is on

Pointing to the tab displays the Workspace page again. When Auto Hide is on, the
Auto Hide icon is horizontal, , (Figure 10). To turn off the Auto Hide function,
choose Auto Hide in the shortcut menu again to clear the check mark, or click the
Auto Hide icon to change it back to vertical, .

34 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Using Workspace Pages

Figure 10. Workspace page horizontal Auto Hide icon

Auto Hide icon

Y To display a Workspace page when multiple workspaces are open

• Click the target workspace tab.

Y To refresh the Chromatogram or Map View for a workspace

1. Open a raw data file in the acquisition queue.

2. In the Workspace Options toolbar, in the Create area, click Refresh, or press the F5 key
on your computer.
The application refreshes the data of the raw data file.

As the acquisition is in progress, the application:

• Updates the associated Chromatogram traces.
• Updates the Map View, Scan Filters, and MSn Browser that have an associated Time
range.
• Automatically applies peak detection to update the retention time of views.
• Updates the associated peak list table.

Note When you apply Refresh, the application refreshes all the views of data that is
currently being acquired. For files that are not in the acquisition mode in a workspace, the
Refresh icon is inactive.

When the acquisition is completed, the Refresh icon is inactive and the F5 key does not
produce any effect.

Y To close a Workspace page

• Right-click the title bar and choose Close.

–or–
• Click the Close icon, .

Thermo Scientific FreeStyle User Guide 35

2 Using the FreeStyle Window
Arranging Views

Arranging Views
You can move or reposition the views to change the layout of the workspace and then save the
modified layout to a template. For information about templates, see Saving and Applying
Layout Templates.

Y To arrange views with the mouse

1. Drag the title bar of the view that you want to move to a second view until the view
arranger tool appears.

2. Do one of the tasks in the following table.

Task Procedure
Move the first view above the Drag the title bar to the up icon, .
second view.
Move the first view below the Drag the title bar to the down icon, .
second view.
Move the first view to the left of Drag the title bar to the left icon, .
the second view.
Move the first view to the right of Drag the title bar to the right icon, .
the second view.
Make both views tabbed. Drag the title bar to the tabs icon, .

The application displays the first view and creates a

tab for the second view.

Figure 11 shows the Spectrum view being dragged to the right of a Chromatogram view,
and Figure 12 shows the end result.

36 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Arranging Views

Figure 11. Spectrum view being dragged to the right of a Chromatogram view

Figure 12. Result of repositioning a Spectrum view to the right of a Chromatogram view

Thermo Scientific FreeStyle User Guide 37

2 Using the FreeStyle Window
Selecting the Columns to Display in a View or Dialog Box with Tabular Data

Selecting the Columns to Display in a View or Dialog Box with

Tabular Data
To minimize the display of infrequently used information, some of the table columns in a
view or dialog box are hidden by default. You can change which columns to display or hide in
a specific table by accessing the table’s Field Chooser dialog box.

Y To open a Field Chooser dialog box and change the column selections

1. Click the Field Chooser icon, , to the left of the table heading row.
2. Do the following:
• To display a column, select its associated check box.
• To hide a column, clear its associated check box.

Figure 13 shows the Field Chooser dialog box for the Chromatogram Ranges view. In this
view, the following columns are hidden by default—Chemical Formula, Mass Tolerance, and
Comment.
Figure 13. Field Chooser dialog box for the Chromatogram Ranges view

Hidden column
Hidden column

Hidden column

You can display or hide table columns in these views and dialog boxes:
• Chromatogram Ranges View
• Spectrum List View
• Status Log View
• Sample Information View
• Peaks List View

38 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Saving and Applying Layout Templates

• Elemental Composition Results View

• Spectrum Ranges Dialog Box
• Fill Down dialog box (see Using the Fill Down Feature)

Saving and Applying Layout Templates

A FreeStyle template contains the settings that define the workspace layout. The template
specifies what views are in a Workspace, how the views are arranged, and what labeling the
views contain. The template also specifies which pages the Info Bar initially displays. For
future use, you can save the template as a new XML file, or save it as the default template.

The FreeStyle application stores all templates in the following folder:

drive:\Users\your name\AppData\Local\Thermo Scientific\FreeStyle\Templates\

Y To save the template as the default template

1. Click the Workspace Options tab to open the Workspace Options toolbar (see
Figure 19).
2. Click Layouts and choose Save As Default from the dropdown menu.

Note You can also choose File > Save As Default.

The application saves the layout as the default template file, DefaultTemplate.xml.

Y To apply the default template

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Layouts and choose Apply Default from the dropdown menu.

Y To name the template and save it as an XML file

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Layouts and choose Save As from the dropdown menu.
The Save Settings dialog box opens.
3. Type a name for the template and click Save.

IMPORTANT Do not navigate to a different folder. The application reads the

template files only from the default template folder.

Thermo Scientific FreeStyle User Guide 39

2 Using the FreeStyle Window
Restoring the Default Settings

Y To apply a saved template

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Layouts and choose Apply from the dropdown menu.
The Browse Templates dialog box opens.
3. Locate and select a template and click Open.

Restoring the Default Settings

The FreeStyle application includes the Default Options Configuration dialog box with a set of
pages containing initial default settings upon installation. You can modify these settings as
needed or restore them to the factory default values.

For information about these settings, see Appendix A, “FreeStyle Default Settings.”

Y To open the Default Options Configuration dialog box

• In the Workspace Options toolbar, click Default Options.

–or–
• Choose File > Default Options.

Y To restore the settings of a particular page to the factory default settings

1. Open the Default Options Configuration dialog box.

2. In the navigation pane, click the page for which you want to restore the default settings.
3. In the upper right corner, click Revert to Factory Default Values.
The application immediately saves these changes. There is no undo. Clicking Cancel does
not return your custom values.

Y To restore all settings for all pages to the factory default settings

1. Open the Default Options Configuration dialog box.

2. In the bottom left corner, click Revert All to Factory Default Values.
3. When prompted to restore all default settings, click OK.
4. When prompted to apply these default settings to open workspaces, click Yes or No.
The application saves these changes. There is no undo. Clicking Cancel does not return
your custom values.

40 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Using the Pointer to Update the Timebase or Rescale a Graph

Using the Pointer to Update the Timebase or Rescale a Graph

Within a Chromatogram View, Spectrum view, or MultiSpectrum view, use the pointer in
three ways:
• To select a retention time or scan number, click the linked chromatogram trace at that
retention time.
• To select a range, drag a line parallel to any axis.
• To select an area, drag in any diagonal direction.

Pointer actions scale the view according to the dimensions of the dragged line or area (see
Table 5).

Note To restore the original scale, right-click the view and choose Reset Scaling.

Table 5. Pointer effects

Pointer action Effect
Drag parallel to the x axis Rescales the graph to the selected x-axis range. The
y-axis range might rescale depending on the selected
normalization display options.
Drag parallel to the y axis Rescales the graph to the selected y-axis range, with
the same x-axis range.
Drag diagonally over the x and y Rescales the graph to the selected x- and y-axes ranges.
axes

In addition, when you select a retention time in a Chromatogram view, the application
automatically synchronizes to display data at that selected RT for the linked Spectrum view, or
the active spectrum in the linked MultiSpectrum view, the Scan Header view, and the Status
Log view in the Workspace.

Adding Text, Graphic, and Structure Annotations to a Graphical View

To add custom text, graphics, and structures to the graphical views, use the features on the
Text and Graphic Annotation toolbar.

Note Except for the Structure icon, the toolset for the Text and Graphic Annotation
toolbar are same for the Chromatogram and Spectrum views. To add labels that the
application generates from the data, use the features on the Labels area of the Display
Options toolbar.

After you select the view and open the Text and Graphic Annotation toolbar, follow these
procedures:
• To place a drawing object behind the plot
• To draw a line

Thermo Scientific FreeStyle User Guide 41

2 Using the FreeStyle Window
Adding Text, Graphic, and Structure Annotations to a Graphical View

• To draw a box or a filled box

• To add a text label
• To edit a text label
• To remove one or more labels
• To save text and structure annotations in the Spectrum view
• To import text and structure annotations
• To annotate a spectral peak with a structure

Y To place a drawing object behind the plot

Click the Behind Graph icon, , before you use the pointer to draw the object.

Y To draw a line

1. To select the line color, click the Line Color icon, , and then select a color from the
graphic.
2. Do one of the following:
• To draw a horizontal line, click the Horizontal Line icon, . and then drag the
pointer horizontally across the view.
• To draw a vertical line, click the Vertical Line icon, , and then drag the pointer
vertically across the view.
• To draw a diagonal line, click the Diagonal Line icon, , and then drag the pointer
diagonally across the view.

Y To draw a box or a filled box

1. To select the border color, click the Line icon, , and then select a color from the
graphic.
2. Click the Box icon, , or the Filled Box icon, .
3. For a Filled Box, click the Fill Color icon, , and then select a color from the graphic.
4. Click the view where you want to place the top left corner of the box, and then drag the
pointer across the view to size the box. You can continue to increase or decrease the box
dimensions until you release the mouse button.

Y To add a text label

1. Click the Add Text icon, , to add a text label.

The Add/Edit Text Annotation dialog box opens.

42 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Adding Text, Graphic, and Structure Annotations to a Graphical View

Figure 14. Add/Edit Text Annotation

2. Type the text in the text box.

You can enter several lines of text by pressing ENTER after each line.
3. To format the text, do the following:
• To display the text in bold and italic, select Bold and Italic.
• To display the text in box or rotate the text, select Boxed or Rotated.
• To specify a text label with a callout line, select Callout.
• To align the text, select Left, Center, or Right.
By default, when there are multiple lines, the text is left aligned. You can change the
alignment to Center or Right.
• To select the text and line color, under Color, click the down-arrow icon.
The default line color is blue and text color is red.
4. To add the text in the Spectrum or Chromatogram view, click the location where you
want to add the text.
The text appears in the selected location.
5. To move the text, point to the text, and when the red box appears, drag the text to the
new location.
Note When you move the text with a callout line, the callout anchor remains at the
original location and only the text moves.

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2 Using the FreeStyle Window
Adding Text, Graphic, and Structure Annotations to a Graphical View

Y To edit a text label

1. Double-click the text label that you want to edit.

The Add/Edit Text Annotation dialog box opens with the text format options.
2. Select the options to edit, and click Edit.

Y To remove one or more labels

Do any of the following:

• To clear selected text labels, click Selected Text, , in the toolbar. Then, drag the
pointer across the text that you want to remove.
• To clear a single label, double-click the text label. Then, remove the text from the
Add/Edit Text Annotation dialog box, and click Edit.
• To clear selected graphic labels, click Selected Graphics, , in the toolbar. Then,
drag the pointer across the graphics that you want to remove.
• To clear all the labels, click All, , in the toolbar.

Y To annotate a spectral peak with a structure

1. Select the Spectrum or MultiSpectrum view.

2. To add a structure annotation, do one of the following:
• In the Tools area, click Structure .
The Structure Annotation dialog box opens and displays the m/z value in the Mass
box.
Figure 15. Structure Annotation

Note By default, the Structure Annotation dialog box displays five rows. The
application automatically adds additional rows to add more annotations.The
structure list added is specific for each spectrum plot and you cannot copy the
structures to a new plot.

44 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Adding Text, Graphic, and Structure Annotations to a Graphical View

3. Click the Mass box and type the m/z value.

4. Click the File Name box, and browse to the location of the .mol file saved in your system.
5. Select the mol file and click Open to import the structure.
6. Specify the appearance of the two-dimensional molecular structure as follows:
• From the Structure Scale dropdown list, select the relative size.
• From the Font Name dropdown list, select the font for the structure’s atoms.
• From the Font Size dropdown list, select the relative size of the text for the structure’s
atoms.
• To bold the text for the structure’s atoms, select the Bold check box.
• To select different colors for the structure’s bonds and atoms, open the respective
color palettes and select the colors.
7. To display the overlapped structures when the structure is bigger than the workspace and
does not appear, select the Draw Overlap Structures check box. By default, the check
box is not selected.
8. Click Apply, and then click OK.
The annotated structure is displayed at the selected mass point in the spectra.
9. To change relative size of the structure directly from the Text and Graphic Annotation
toolbar, click and move the Scale slider.
Figure 16. Structure Scale Slider

10. To change a structure’s position, drag and drop the structure within the view.
The structure changes only the position but remains linked to the original mass value
until you edit the mass.
11. To clear the structure annotations, right-click the Spectrum view and choose Clear
Structure Annotations.
Choosing Clear Structure Annotations removes the annotations for the Spectrum view
and clears the list from the Structures Annotation dialog box.

Note The application does not import the annotations to a copy of the spectrum
within the workspace, but when you copy the spectrum to the Clipboard and paste it
into other applications (such as PowerPoint™, Paint™, and so on), the application also
copies the annotations to the new file.

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2 Using the FreeStyle Window
Copying, Exporting, and Printing Graphical Images and Tabular Data

Y To save text and structure annotations in the Spectrum view

1. Select the Spectrum or MultiSpectrum view.

2. In the Save area, click .
The Save Settings dialog box opens.
3. Select the file to save the annotation, and click Save.
The XML file saves the following information:
• Text annotations
• Structure annotations
• Boxed label
• Bold and Italic styles
• Callout link
• Rotated format
• Text color
• Line color
• Alignment

Y To import text and structure annotations

1. In the Save area, click .

The Browse Templates dialog box opens.
2. Select the annotation XML file you want to import to the workspace.
3. Click Open.
The application imports the text and structure annotation to the Spectrum view.

Copying, Exporting, and Printing Graphical Images and Tabular Data

You can copy graphical views and tabular data to the Clipboard, send the contents of a
Workspace or view to a printer, or export the contents of a Workspace or view to a CSV or an
EMF file.

Follow the instructions in these topics:

• Copying an Image of a Graphical View to the Clipboard
• Copying Tabular Data to the Clipboard
• Exporting or Printing the Contents of a View or Workspace

46 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Copying, Exporting, and Printing Graphical Images and Tabular Data

Copying an Image of a Graphical View to the Clipboard

You can copy an image of the following views or floating window to paste into other
Microsoft Office applications: Chromatogram view, Spectrum view, MultiSpectrum view, and
Isotope Simulation window.

Y To copy an image of a graphical view or floating window to the Clipboard

Right-click the view or window and choose Copy To Clipboard.

Copying Tabular Data to the Clipboard

You can copy the contents of the eight report views (see Reports) to the Clipboard.

Y To copy all the contents of a report view to the Clipboard

1. Open a report view.

2. Click the view.
3. Press CTRL+C.
The application copies the report contents to the Clipboard.

Exporting or Printing the Contents of a View or Workspace

To print, copy, or export the contents of a view or workspace, use commands on the Exports
menu on the Workspace Options toolbar.

Clicking either command opens the Copy to Clipboard/Export dialog box where you select
the export type, the output size for a Clipboard image, and the number of pages to print.

Y To export, copy, or print the contents of a view or workspace

1. Do one of the following:

• To export the contents of a view, click the view, click Exports, and choose Export
Selection As from the dropdown menu.
• To export the contents of a workspace, click Exports, and choose Export Workspace
As from the dropdown menu.
The Copy to Clipboard/Export dialog box opens (Figure 17).

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2 Using the FreeStyle Window
Copying, Exporting, and Printing Graphical Images and Tabular Data

Figure 17. Copy to Clipboard/Export dialog box

2. In the Export Type area, select one of these options:

• To export the data in tabular format to a CSV file, select the To CSV File option.
Then, go to step 4.
• To send an enhanced metafile image to the Clipboard, select the To Clipboard in
EMF option. Then, in the Output Size (For Clipboard) area, use the Width (in.) and
Height (in.) boxes to specify the size of the image.
• To send the printing information to the selected printer, select the Print option.
Then, select one of the following:
– To print the graphical views in a workspace on one page, select the One Page
option.
– To print the graphical views in a workspace on separate pages or all the pages in a
report view, select the Separate Page option.
3. Click OK.
4. Depending on the export type, do the following:
• For the To CSV File option, in the Export Data dialog box, select the folder location,
name the file, and click Save.
• For the To Clipboard in EMF option, paste the image from the Clipboard to the
appropriate document.
• For the Print option, in the Print dialog box, select the printer, the printing
preferences, the page range, and the number of copies. Then, click Print.

Tip To create a PDF file, select the Adobe PDF printer.

48 FreeStyle User Guide Thermo Scientific

 2 Using the FreeStyle Window
Setting FreeStyle as the Default Data Visualization Application

Setting FreeStyle as the Default Data Visualization Application

Currently, Thermo Fisher Scientific provides two data visualization applications: Xcalibur and
FreeStyle.

Y To specify the FreeStyle application as the default data visualization application

1. Open Windows Explorer and browse to a raw data file.

2. Right-click the file, and then choose Open With > FreeStyle Version (Figure 18).
Figure 18. Choosing FreeStyle as the default data visualization application

Toolbars
Use the FreeStyle toolbar features to display views and to perform functions. The FreeStyle
window has these toolbars:
• Workspace Options Toolbar
• Workspace Processing Toolbar
• Display Options Toolbar
• Zoom Options Toolbar
• Text and Graphic Annotation Toolbar
• Sequence Toolbar (in the Reviewing Chromatographic Data chapter)

Tip The FreeStyle window displays only the toolbars and toolbar features that are
appropriate for the selected view. For example, the Peak Detection features in the
Workspace Processing toolbar are available only when a Chromatogram view is selected,
and the Library Search icons in this toolbar are available only when a Spectrum or
MultiSpectrum view is selected.

The Sequence toolbar appears when you select a file on the Sequence File page.

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2 Using the FreeStyle Window
Toolbars

Workspace Options Toolbar

Use the features in the Workspace Options toolbar to create, customize, and refresh
workspaces.

Y To display the Workspace Options toolbar

Click the Workspace Options tab.

Figure 19 shows the Workspace Options toolbar features that are available when you select a
Chromatogram view.
Figure 19. Workspace Options toolbar for the Chromatogram view
Workspace Options tab

Depending on the active view, these additional features—Ranges, Multi Spectrum, CV Plot,
and Write to .RAW—become available as follows:
• Selecting a Spectrum view or a MultiSpectrum view makes the Ranges and Spectrum
commands available on the Spectrum menu.
• Opening a raw data file (.raw) with FAIMS data makes the CV Plot icon available.
• Selecting a Chromatogram view, a Spectrum view, or a MultiSpectrum view makes the
Exports – Write to .RAW command available.

Table 6 describes the menus and icons in the Workspace Options toolbar, from left to right.
Table 6. Workspace Options toolbar commands (Sheet 1 of 4)
Command Description
Create area
New Workspace Displays the Open Raw File dialog box. You create a workspace by opening a raw data file or a
sequence of raw data files. A workspace can display selected information from one or more raw
data files. The workspace tab includes the file name of the raw data file when the Workspace
displays information from only one raw data file.
Create Sequence Opens the Create Sequence dialog box. Use the Create Sequence dialog box to build a
sequence from raw data files.
Refresh Updates a view that is showing a chromatogram or a map from a raw data file that is currently
being acquired. The data system expands the display range to show the full range of the data
acquired.

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Toolbars

Table 6. Workspace Options toolbar commands (Sheet 2 of 4)

Command Description
Workspace area
Chromatogram menu

Ranges Opens the Chromatogram Ranges view where you can specify the chromatogram traces to
display.
Insert Trace Adds another chromatogram trace within the selected Chromatogram view. Use the
Chromatogram Ranges view or the Auto Filter icon to select the chromatograms of interest.
Insert View Adds a Chromatogram view to the Workspace.
Spectrum menu
Ranges Available when a Spectrum view or a MultiSpectrum view is selected and these views do not
include a library spectrum.

Displays the Spectrum Ranges dialog box, where you select the spectral trace or traces to
display in a Spectrum view or MultiSpectrum view, respectively.
Multi Spectrum Displays the MultiSpectrum view, where you can group multiple spectra together in one view.
Insert View Displays a Spectrum view, where you view the spectrum for the chosen retention time (RT)
and scan number.
CV Plot Compensation voltages (CV) plot is available for raw data files with FAIMS data.

Opens the CV Plot Traces dialog box, where you select the CV plots of interest.
Map View menu
Ranges Opens the Map Ranges dialog box to specify ranges for a map view.
Insert View Adds a Map View of the active chromatogram trace.

Displays a map of the currently selected raw file in the map view where you can view the
retention time (RT) and m/z values.
Data Analytics menu
Ranges Opens the Data Analytics Ranges dialog box to specify the filename, detector type, trace type,
and filter for the view.
Insert View Inserts a Data Analytics View in the workspace.
Layouts menu
Save As Saves the current layout as an XML template file. See Saving and Applying Layout Templates.
Save As Default Saves the current layout as the default template file, DefaultTemplate.xml. See Saving and
Applying Layout Templates.
Apply Opens the DataBrowser Templates dialog box, where you select a previously saved layout
template. See Saving and Applying Layout Templates.

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Toolbars

Table 6. Workspace Options toolbar commands (Sheet 3 of 4)

Command Description
Apply Default Applies the default layout template, DefaultTemplate.xml. See Saving and Applying Layout
Templates.
Exports menu
Write to .RAW For a chromatogram trace, saves the MS scans for the displayed time range of the selected
chromatogram trace to a new raw data file and appends _FS to the file name (see Exporting
Scans for a Filtered Chromatogram to a New Raw Data File). The new raw data file includes
only the MS scans for the filtered chromatogram.

For a spectrum or multispectrum plot, saves the selected spectrum to a new raw data file,
appends Scan [scan number] to the file name (see Exporting a Selected Spectrum to a New
Raw Data File), and records the action in the Audit Trail.
Note When the raw data file contains Advanced Peak Determination (APD) data—such as
species ID, monoisotopic mass, and top peak in the cluster—the exported file does not
include the APD data.
Export Opens the Copy to Clipboard/Export dialog box, where you specify settings to print all the
Workspace As workspace pages, export them to a CSV file, or copy them to the Clipboard (see Exporting or
Printing the Contents of a View or Workspace).
Export Selection Opens the Copy to Clipboard/Export dialog box, where you specify settings to print the
As selected view, export it to a CSV file, or copy it to the Clipboard (see Exporting or Printing the
Contents of a View or Workspace).
Filter icons
Auto Filter Opens the To specify the maximum number of chromatogram traces to display, where you can
specify the filters to use to repopulate an existing Chromatogram view with filtered plots.

Depending on the selections you made in the Auto Filter dialog box, the Chromatogram View
is populated with the selected scan filters. The default selected scan filters are dependent on
the number specified on the Workspace Options page of the Default Options Configuration
dialog box. Also populates the Chromatogram Ranges view with the specified number of scan
filters. See Adding Chromatogram Traces with the Auto Filter Feature.
Scan Filters Opens the Scan Filters page in the Info Bar (see Using the Scan Filters Page to Display a
Filtered Chromatogram).
Reports menu
Spectrum List Displays the Spectrum List view, where you view spectral peak information in a table. Click
the chromatogram trace to display the spectrum list for the chosen retention time and scan
number.
File Header Displays the File Header view, where you view information from the acquisition sequence, the
autosampler, and the mass spectrometer.

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 2 Using the FreeStyle Window
Toolbars

Table 6. Workspace Options toolbar commands (Sheet 4 of 4)

Command Description
Scan Header Displays the Scan Header view, where you view the scan header of the active raw data file.
Click the chromatogram trace to display the scan header for the chosen retention time and
scan number. See Scan Headers and Scan Header Abbreviations.
Instrument Displays the Instrument Method view, where you view the instrument method parameters
Method that the instrument used to obtain the raw data file.
Status Log Displays the Status Log view, where you view instrument readback parameters. Click the
chromatogram trace to display the status log for a specific retention time and scan number.
Tune Method Displays the Tune Method view, where you view the tune method parameters that the
instrument used to obtain the raw data file.
Sample Displays the Sample Information view, where you view sample-specific information.
Information
Error Log Displays the Error Log view, where you view a list of error messages generated during data
acquisition.
Configuration area
Default Options Opens the Default Options Configuration dialog box, where you specify the default settings
for the application (see FreeStyle Default Settings).

Workspace Processing Toolbar

The available features in the Workspace Processing toolbar depend on whether you select a
Chromatogram view or a Spectrum (or MultiSpectrum) view.

Note The Isotope Simulation and Library Manager features are available for both
Chromatogram and Spectrum views.

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Toolbars

For information about the Workspace Processing toolbar, see these topics:
• Chromatogram – Workspace Processing Toolbar
• Spectrum – Workspace Processing Toolbar
• Map View – Workspace Options Toolbar
• Data Analytics View – Workspace Options Toolbar

Display Options Toolbar

For information about specific features on the Display Options toolbar, see these topics:
• Chromatogram – Display Options Toolbar
• Spectrum – Display Options Toolbar
• Spectrum List – Display Options Toolbar
• Map View – Display Options Toolbar
• Data Analytics View – Display Options Toolbar

Zoom Options Toolbar

Use the features in the Zoom Options toolbar to adjust the display of chromatograms or
spectra.

Note The Zoom Options toolbar is available when a Chromatogram view, a Spectrum
view, a Map view, or a MultiSpectrum view is selected.

Figure 20 shows the Zoom Options toolbar, and Table 7 describes the toolbar features.
Figure 20. Zoom Options toolbar

Table 7. Zoom Options toolbar icons (Sheet 1 of 2)

Icon Description
Reset Restores the data display to the full range of the x axis and y axis.

Zoom In Y Zooms in on the y axis by a factor of two from the current

baseline to show more detail. For example, you can change the
y-axis range from 0–100 to 0–50.

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 2 Using the FreeStyle Window
Toolbars

Table 7. Zoom Options toolbar icons (Sheet 2 of 2)

Icon Description
Zoom Out Y Zooms out from the y axis by a factor of two from the current
baseline to show more data. For example, you can change the
y-axis range from 0–25 to 0–50.
Zoom In X Zooms in on the x axis by a factor of two from the center to show
more detail. For example, change the x-axis range from 0–20 to
5–15.
Note The first time you click Zoom In X, the application
zooms in on the full time range of the active chromatogram
plot. The plot’s time range depends on the selected scan filter
and can be shorter than the data acquisition time for the raw
data file.

A horizontal scrollbar automatically appears below the x axis of

the graphical view when you zoom in.
Zoom Out X Zooms out from the x axis by a factor of two from the center to
show more data. For example, change the x-axis range from 7.5–
12.5 to 5–15.

Text and Graphic Annotation Toolbar

Use the features in the Text and Graphic Annotation toolbar to annotate chromatograms and
spectra with text, lines, and boxes. You can also select color, text alignment, and various text
options.

For information about working with the Text and Graphic Annotation toolbar, see Adding
Text, Graphic, and Structure Annotations to a Graphical View.

Note The Text and Graphic Annotation toolbar is available when you select a
Chromatogram view, a Spectrum view, or a MultiSpectrum view.

Figure 21 shows the Text and Graphic Annotation toolbar, and Table 8 describes the toolbar
features.
Figure 21. Text and Graphic Annotation toolbar
Text and Graphic Annotation tab

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Toolbars

Table 8. Text and Graphic Annotation toolbar commands

Command Description
Drawing area
Add Text Annotates with a text label (see To add a text label).
Horizontal Line Draws a horizontal line when you drag the mouse pointer
horizontally.
Vertical Line Draws a vertical line when you drag the mouse pointer vertically.
Diagonal Line Draws a diagonal line when you drag the mouse pointer
diagonally.
Box Draws an empty box when you drag the mouse pointer
diagonally.
Filled Box Draws a box filled with the fill color when you drag the mouse
pointer diagonally.
Modes area
Line Color Opens the color box to select the line color.

Fill Color Opens the color box to select the fill color and the text color.

Behind Graph Places text, lines, and boxes behind the chromatogram traces or
mass spectra.
Clear area
Selected Text Deletes text annotations when you drag the pointer over them.

Selected Deletes graphic annotations when you drag the pointer over
Graphics them.
All Deletes all text and graphic annotations.

Structure area
Structure Displays the structure annotations of the selected spectrum.

Scale Adjusts the scale of the structure annotations.

Save area
Save Saves the text annotation for the selected trace in XML format.
Annotations
Apply Imports the text annotation to the selected trace.
Annotations

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 2 Using the FreeStyle Window
Views

Views
A view is a pane on a Workspace page. Use the available views to display results and to open
lists for entering parameters or selecting options. You can drag views to arrange them within
the workspace of the application (see Arranging Views).

The FreeStyle window displays these individual views (by type in alphabetical order) inside
the Workspace page. In addition, Table 9 lists related topics for detailed information and the
type of data the view contains for export.
Table 9. Views (Sheet 1 of 2)
View Topic Copy as
Primary
Chromatogram Chromatogram View Image
Chromatogram Ranges Chromatogram Ranges View Tabular text
MultiSpectrum MultiSpectrum View Image
Spectrum Spectrum View Image
Map View Working with the Map View Image
Data Analytics View Adding Data Analytics View to the Image
Workspace
Reports
Error Log Error Log View Tabular text
File Header File Header View Tabular text
Instrument Method Instrument Method View Tabular text
Sample Information Sample Information View Tabular text
Scan Header Scan Header View Tabular text
Spectrum List Spectrum List View Tabular text
Status Log Status Log View Tabular text
Tune Method Status Log View Tabular text
Data processing – Elemental composition
Elemental Composition Elemental Composition Results View Tabular text
Results
Data processing – Chromatographic peak detection and integration
Peaks List Peaks List View Tabular text
Data processing – Library searches
Chemical Structure Chemical Structure view from mzVault or Image
NIST

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Views

Table 9. Views (Sheet 2 of 2)

View Topic Copy as
Compounds list mzVault Search Results View Tabular text
Compounds list NIST Search Results View Tabular text
Data processing – Xtract deconvolution
Deconvolved Spectrum Deconvolved Spectrum View Image
Xtract Results Xtract Results View Tabular text

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 2 Using the FreeStyle Window
Info Bar Pages

Info Bar Pages

On the left of the FreeStyle window, use the pages of the Info Bar to select detector, sequence,
or filter options. You can also set the parameters for peak detection, isotope simulation,
elemental composition, peptide fragments analysis, searching mass spectrum libraries, or
applying the Xtract deconvolution algorithm.

The following pages appear in the Info Bar:

• Chromatogram-range pages: Only one of these two pages is available at any time.
– Detector Type Page
– Trace Type Page
• Scan Filters Page
• Elemental Composition Page
• Isotope Simulation Page
• Modifying an mzVault Search from the mzVault Search Page
• MSn Browser Page
• Peptide Fragments Info Bar
• Modifying a NIST Search from the NIST Search Page
• Peak Detection Page
– Avalon Peak Detection Page
– Genesis Peak Detection Page
– ICIS Peak Detection Page
– PPD Peak Detection Page
• Sequence File Page
• Xtract Page for a Selected Spectrum

To rearrange or hide the Info Bar pages, follow the procedures in these topics:
• Rearranging the Info Bar Pages
• Hiding the Info Bar Pages
• Docking or Floating the Info Bar Pages

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Info Bar Pages

Rearranging the Info Bar Pages

Y To move an Info Bar page

Drag its tab left or right.

Hiding the Info Bar Pages

Y To hide an Info Bar page

Right-click its tab and choose Auto Hide.

Y To hide all the Info Bar pages

Right-click the Info Bar title bar and choose Auto Hide.

Docking or Floating the Info Bar Pages

Do one of the following:
• Right-click the Info Bar title bar and choose Floating to detach the Info Bar pages into a
floating window.
• Right-click the Info Bar title bar and choose Dockable to return the Info Bar pages back
to their default position in the FreeStyle window.

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 3

Reviewing Chromatographic Data

To review the chromatographic data, follow the procedures in these topics.

Contents
• Adding Chromatogram Views
• Closing Chromatogram Views
• Adding Chromatogram Traces
• Deleting Chromatogram Traces
• Displaying Mass Defect Filtered Chromatogram Traces
• Setting up Instrument Status Traces
• Displaying an EIC Trace
• Using the Scan Filters Page to Display a Filtered Chromatogram
• Setting Up the Display Options for a Chromatogram Trace
• Changing the Zoom Level of a Chromatogram
• Automatically Detecting and Integrating Chromatographic Peaks
• Selecting the Manual Noise Region for the Genesis and ICIS Algorithms
• Manually Adding, Undoing, and Deleting Chromatographic Peaks
• Importing Components from a Processing Method
• Exporting Scans for a Filtered Chromatogram to a New Raw Data File
• Working with Sequences
• Chromatogram-specific Toolbars
• Chromatogram-Specific Views
• Chromatogram-specific Pages in the Info Bar

Note For information about adding images and annotations to and copying an image of a
Chromatogram view, see Using the FreeStyle Window.

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3 Reviewing Chromatographic Data
Adding Chromatogram Views

Adding Chromatogram Views

You can add multiple Chromatogram views to a Workspace.

Y To add a Chromatogram view

In the Workspace Options toolbar, click Chromatogram View.

Result:
• When the Workspace includes one or more Chromatogram views, a copy of the
selected or active Chromatogram view (as indicated by the tilde in its title bar)
appears.
• When the Workspace does not include a Chromatogram view, a new Chromatogram
view appears with the default chromatogram (based on the raw data file) and display
settings. The chromatogram trace is linked to the MSn Browser page and any other
opened view.

Note When the Workspace does not include a Chromatogram view, you can also add the
first Chromatogram view by clicking Auto Filter on the Workspace Options toolbar (see
Adding Chromatogram Traces with the Auto Filter Feature).

Closing Chromatogram Views

A Workspace must include at least one of the following views: a Spectrum view, a
Chromatogram view, a Map View, or a report view. When you close all the Chromatogram
views, the remaining views and the MSn Browser page retain information based on the last
selected chromatogram trace.

Y To close a Chromatogram view

Click the Close icon.

The effect of closing a Chromatogram view depends on whether it is the only
Chromatogram view in the Workspace, whether any other views are open, and the linkage
state of any spectra (Table 10).

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 3 Reviewing Chromatographic Data
Adding Chromatogram Traces

Table 10. Effect of closing a Chromatogram view

When you close … The application does the following …
The last • Releases any spectrum that is linked to the chromatogram.
Chromatogram view in The text appended to the Spectrum view’s title bar changes
the Workspace from CxTy to Released.
• Closes the Chromatogram Ranges view.
• Closes the CV Plot view.
• Closes the Workspace if the Chromatogram view was the last
view in the Workspace.
One of the • Makes any spectrum that was linked to a chromatogram in
Chromatogram views the closed view available for linking to another
in the Workspace chromatogram.
– The text appended to the Spectrum view’s title bar
changes from CxTy to ‘Select Trace to Link’.
– Clicking another Chromatogram view automatically links
the spectrum to the selected chromatogram.
• Links an opened Chromatogram Ranges view to the active
Chromatogram view.
• Closes the CV Plot.

Adding Chromatogram Traces

The factory default Workspace includes a Chromatogram view with a single trace and a
Spectrum view that is linked to the trace (see Opening Raw Data Files or Sequence Files). You
can add multiple chromatogram traces to the Chromatogram view and multiple
Chromatogram views to the Workspace.

By default, when you add multiple traces to a Chromatogram view, the traces are stacked. As
you add more traces, each successive trace appears in a different color until the view contains
eight traces. The color order is as follows: (1) black, (2) brick-red, (3) green, (4) blue, (5) light
orange, (6) magenta, (7) blue-green, and (8) gray. As you add more traces, this color pattern
repeats.

The application supports the following types of chromatogram traces for MS data (Table 11).
To select the TIC, BPC, Mass Range, Base Peak, Neutral Fragment, or Mass Defect Filter
trace type, use the Chromatogram Ranges view.

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Adding Chromatogram Traces

Table 11. Supported chromatogram traces

Trace type Description
Total ion current (TIC) A trace of the signal of all ions combined as a function of the
retention time or scan number.
Base peak (BPC) A trace of the most intense ion signal as a function of the
retention time or scan number.
Mass range A trace of the ion signal of all ions in one or more mass ranges as
a function of the retention time or scan number.
Extracted ion (EIC or A trace of the signal of a single ion as a function of the retention
XIC) time or scan number.
Neutral fragment A trace of the ion signal of all ions that produce a specific neutral
fragment as a function of the retention time or scan number.
Mass Defect Filter A trace of the ion signal of all ions in one or more mass defect
ranges as a function of the retention time or scan number.

Follow the procedures in these topics as needed:

• Adding Chromatogram Traces Manually
• Adding Chromatogram Traces with the Auto Filter Feature

Adding Chromatogram Traces Manually

To manually add chromatogram traces to a Chromatogram view, follow these procedures as
needed:
• To add a chromatogram trace by using the Chromatogram menu
• To add a chromatogram trace by using the shortcut menu command
• To add chromatogram traces by using the Chromatogram Ranges view
• To add a chromatogram trace from a raw data file in a sequence list

Note To automatically populate a Chromatogram view with traces from multiple scan
filters, see the next topic Adding Chromatogram Traces with the Auto Filter Feature.

Y To add a chromatogram trace by using the Chromatogram menu

1. Click a Chromatogram view or a specific trace in the Chromatogram view to select it.
2. Click the Workspace Options tab to display the Workspace Options toolbar.
3. Click Chromatogram and choose Insert View from the dropdown menu.
A copy of the selected chromatogram appears at the bottom of the view.

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 3 Reviewing Chromatographic Data
Adding Chromatogram Traces

Y To add a chromatogram trace by using the shortcut menu command

Right-click a Chromatogram view and choose Insert Chromatogram.

A copy of the selected chromatogram appears at the bottom of the view.

Y To add chromatogram traces by using the Chromatogram Ranges view

1. In the Workspace Options toolbar, choose Chromatogram > Ranges.

The Chromatogram Ranges dialog box linked to the active or selected Chromatogram
view opens.
2. In the last row, select the check box in the Display column.
The application populates the row with a copy of the currently selected row in the list,
and a duplicate chromatogram trace appears in the Chromatogram view. To fill multiple
rows, see Using the Fill Down Feature. To populate the Chromatogram Ranges view with
ranges from a spreadsheet file, see To specify the ranges of the chromatogram traces by
using data in a spreadsheet file.

Y To add a chromatogram trace from a raw data file in a sequence list

1. Open an existing sequence file (SLD), or create a temporary sequence list by clicking
Create Sequence in the Workspace Options toolbar and selecting a set of raw data files.
2. To display the Sequence toolbar, click the Sequence File tab in the Info Bar.
3. Click New Trace.
4. Click a raw data file in the sequence.
The application adds the raw data file to the chromatogram ranges list and displays the
new chromatogram trace in the selected Chromatogram View.

Adding Chromatogram Traces with the Auto Filter Feature

Use the To specify the maximum number of chromatogram traces to display to specify the
filters to use to repopulate an existing Chromatogram view with these possible characteristics:
• A plot showing the chromatogram without any scan filters
• A plot showing the chromatogram using generic scan filters, such as MS, MSn, CID,
ETD, HCD, or UVPD.
• Plots for specific scan filters applied to the chromatogram up to the number of scan filters
in the data file or the maximum number specified on the Workspace Options Page,
whichever is fewer

Note To manually add traces to a Chromatogram view, see Adding Chromatogram Traces.

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Adding Chromatogram Traces

Y To display a filtered chromatogram

1. Open the raw data file of interest.

2. In the Workspace Options toolbar, in the Filter area, click the Auto Filter icon, .
Figure 22. Auto Filter dialog box

Table 12. Auto Filter dialog box parameters

Parameter Description
Empty Filter Applies no filter.
Generic Filters Lists filters such as MS, MSn (2–10), CID, ETD, HCD, or
UVPD.
Specific Filters Lists all specific filters or only those that match the filter
parameters specified in the Subset box.

When you select the Specific Filters option, clear the Empty
Filter and Generic Filters options, and select the Merge – CV
option, the application ignores the CV values and merges the
scan filters for which all other values are identical in the
FAIMS raw data file.
Subset Specifies scan filter parameters. For descriptions of valid scan
filters, see Appendix B, “Scan Filters and Scan Headers.”
CV Merges the filters based on the compound names.
Use Time Limits Specifies a range (in minutes) to limit the filtered scans.
Range: based on range in the raw data file
Help Opens the Help to the Auto Filter dialog box page (this
page).

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 3 Reviewing Chromatographic Data
Adding Chromatogram Traces

3. In the Auto Filter dialog box, select the type of filter options that you want to use.
The application lists all filters that match the filter criteria.
4. (Optional) In the Filter list, clear the check boxes for any individual filters that you want
to exclude.
5. Click Apply to apply the specified filters to the current Chromatogram view, or click OK
to apply the filters and close the dialog box.
• When you select the Specific Filters option, clear the Empty Filter and Generic Filters
options, and select the CV option, the returned scan filters are merged based on the
precursor mass in the FAIMS raw data file.
• When you select other combinations of filter parameters, the application plots the
number of traces as the number of scan filters seen on the Scan Filters Page on the Info
Bar with maximum limit as set on the Workspace Options Page on the Default Options
Configuration.
This figure shows the result of applying filters to the drugx_01.raw file.
Figure 23. Chromatogram view for a data file with five scan filters and one unfiltered TIC

Y To specify the maximum number of chromatogram traces to display

1. Open the Workspace Options Page.

2. In the #Number of Auto Filter box, type an integer from 1 to 500.
The default maximum number of traces is 8.

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3 Reviewing Chromatographic Data
Deleting Chromatogram Traces

Deleting Chromatogram Traces

To delete chromatogram traces or undo the most recent deletion of a chromatogram trace in a
Chromatogram view, follow these procedures as needed:
• To delete a trace by clicking the delete icon
• To delete a trace by using the Chromatogram Ranges view
• To undo the most recent deletion of a chromatogram trace

Note Deleting a chromatogram trace makes any spectrum that is linked to it available for
linking to another trace—that is, the text appended to the Spectrum view’s title bar
changes from CxYy to ‘Select Trace to Link’. Deleting all the traces in a chromatogram
view closes the view.

Y To delete a trace by clicking the delete icon

1. In the Chromatogram view, point to the name of the trace that you want to delete.
A delete icon appears to the right of the trace.
This figure shows a Chromatogram view with three stacked traces. The pointer is on the
delete icon, , for the first trace.
Figure 24. Chromatogram view with three stacked traces

Delete
icon

2. Click the delete icon, , to delete the trace.

Y To delete a trace by using the Chromatogram Ranges view

• Clear the trace’s associated check box in the Display column.

–or–
a. Select the row, and then press the Delete key.

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b. At the prompt, click Yes.

Y To undo the most recent deletion of a chromatogram trace

Right-click the Chromatogram view and choose Undo Delete Chromatogram.

Defining a Chromatogram Trace from the Chromatogram Ranges View

The following parameters define the range for a chromatogram trace—raw data file, detector,
scan filter, trace type, and mass range for the Mass Range trace type. After you open a raw data
file (see To open a raw data file or a sequence file), you use the Chromatogram Ranges view to
select the parameter settings for each chromatogram plot.

You can copy the rows in the Chromatogram Ranges view to the Clipboard, and you can copy
the data from a CSV file into the Chromatogram Ranges view.

To define the chromatogram ranges, follow the procedures in these topics as needed:
• Manually Defining Chromatogram Ranges
• Defining Chromatogram Ranges by Using a Spreadsheet File

Manually Defining Chromatogram Ranges

To specify the range for each chromatogram trace by manually entering the parameter settings
in the Chromatogram Ranges view, follow these procedures:
• To manually specify the ranges of the chromatogram traces
• To specify smoothing for a plot
• To specify reference (fixed) plots

For information about setting a delay time or using the plot operators, see Chromatogram
Ranges View. For information about adding multiple rows with the same settings, see Using
the Fill Down Feature.

Y To manually specify the ranges of the chromatogram traces

1. In the Workspace, select the Chromatogram view to define.

2. In the Workspace Options toolbar, choose Chromatogram > Ranges.
The Chromatogram Ranges view opens. Its title bar specifies the linked Chromatogram
view.

Note Each row in the Chromatogram Ranges view defines one trace in the selected
Chromatogram view.

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3. For each trace to display in the Chromatogram view, select another check box in the
Display column.
4. In the File Name column, select the raw data file for each trace.
The available detector types depends on the experimental data in the raw data file.
5. In the Detector Type column, select the detector.
Other selections, such as Filter and Trace Type, depend on the selected detector.

Detector Data type

MS Mass spectrometry data
The raw data files generated by an LC/MS or LC/MS/MS
experiment typically contain multiple scan filters to optimize data
collection. Use the remaining columns to define the trace range.
MS Trending Instrument status readings for the mass spectrometer
Use the Trace Type column to specify the specific status readback.
For more information, see Setting up Instrument Status Traces.
UV Data from an analog detector
Use the Trace Type column to define the trace.
PDA Wavelength scan data from a photodiode array detector
A/D Card Data from an analog-to-digital converter

6. For MS data, select a scan filter from the Filter list or from the Scan Filter page.

Tip To display the Scan Filter page in the Info Bar, in the Workspace Options toolbar,
click Scan Filters.

7. In the Trace Type column, do one of the following:

• For UV data, select the channel.
• For PDA data, select the trace type to determine the signal intensity for each
measured time point.

Trace type Effect

Total Scan Plots the total signal.
Wavelength Range Plots the total signal for the wavelength range that you
specify in the Ranges box. Continue at step 10.
Spectrum Maximum Plots the maximum signal.

• For A/D data, select the card.

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• For MS data, select TIC, Mass Range, Base Peak, Neutral Fragment, or Mass
Defect Filter.
Note Entering a chemical formula or peptide sequence in the Chemical Formula
column automatically changes the trace type to Mass Range and populates the
Range column with a calculated m/z value.

• For MS Trending data, select the status readback.

8. For MS data and the Mass Range, Base Peak, or Neutral Fragment trace type, specify the
mass range by doing one of the following:
• In the Ranges box, type the mass range. Use a hyphen to define the mass range.
–or–
a. In the Chemical Formula column, click the table cell to open a dialog box.

b. Select Chemical Formula or Peptide.

c. Type a chemical formula or a peptide sequence.
Use the IUPAC nomenclature (periodic table symbols) for chemical formulas. Use
the one-letter amino acid abbreviations for peptide sequences. See Appendix C,
“One- and Three-Letter Abbreviations for Amino Acid Residues.”
d. Select an adduct species and a charge.
e. Click Apply.
The application populates the Chemical Formula column with the specified formula
or peptide sequence. It also populates these columns:
• Trace Type: Mass Range
• Ranges: m/z value of specified ion
• Mass Tolerance: 0.05 amu for ion trap data or 5 ppm for FTMS data
The EIC trace for the specified mass range appears in the Chromatogram view.
9. For the Mass Range trace type, in the Mass Tolerance column, specify the mass tolerance
and the units.
10. For PDA data and the Wavelength Range trace type, in the Ranges box, type the
wavelength range. Use a hyphen to define the wavelength range.

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Y To specify smoothing for a plot

1. In the Smoothing column, select one of these smoothing algorithms: None, Gaussian, or
Moving Mean.
2. For Gaussian and Moving Mean smoothing, select the smoothing level from a list of odd
integers: 3, 5, 7, 9, and 11.
The smoothing level increases as the selected value increases.

Y To specify reference (fixed) plots

Note A reference plot is a fixed plot—that is, the application does not replace a
reference plot when you compare raw data files in a sequence set.

Select the check box in the Reference column.

Defining Chromatogram Ranges by Using a Spreadsheet File

For information about editing the columns in the Chromatogram Ranges view, see
Chromatogram Ranges View.

To add defined chromatograms by using the data in a spreadsheet file, follow this procedure.

Y To specify the ranges of the chromatogram traces by using data in a spreadsheet file

1. Copy rows from the Chromatogram Ranges view to a spreadsheet file as follows:
a. In the Workspace Options toolbar, choose Chromatogram > Ranges.
b. Click in the area to the left of a row to select the row to copy.
Figure 25. Example pointer location to copy a row

Click this area.

Tip Use the CTRL key to select non-adjacent rows. Use the SHIFT key for
adjacent rows, or drag the pointer across a group of rows.

c. Press CTRL+C.
d. Open the Excel file and select the starting cell that you want to paste into.

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e. Press CTRL+V, and then save the file.

In the Excel file, the pasted rows appear to the right and down from the starting cell.

Tip To copy the entire contents of the Chromatogram Ranges view to a CSV file,
click Selection As in the Workspace Options toolbar.

2. Open the CSV file and edit its contents. Do not change the column format.
3. Paste rows from a spreadsheet file into the Chromatogram Ranges view, as follows:
a. In the spreadsheet file, select the rows that you want to copy. Make sure that this
block is highlighted and has the same number of columns as in the Chromatogram
Ranges view.
b. Click Copy or press CTRL+C.
c. In the Chromatogram Ranges view, select the row where you want to paste the data.
d. Press CTRL+V.
The FreeStyle application overwrites the selected row and the rows below it with the
copied rows.

Displaying Mass Defect Filtered Chromatogram Traces

Mass defect is the difference between the accurate mass of an element (or compound) and its
closest integer value (nominal mass). It might be positive (larger than the nominal mass) or
negative (smaller than the nominal mass). To use MDF, you must use high mass accuracy data
from high resolution analyses.

The Multiple Mass Defect Filter (MMDF) in the FreeStyle application is a data filtering
method that removes ions with the mass defect outside the defined mass defect window. The
filtering of ions can be based on the mass defect of the parent compound and its
modifications. In the FreeStyle application, you can combine the results from multiple mass
defect filters.

Mass defect filter is applied to remove the ions that fall outside the defined molecular weight
range, and those that lies within the range but exceeds the expected mass defect range. This
method lets you to focus on the analysis of potential drug metabolites.

Figure 26 shows an example of the Mass Defect Filtered (MDF) Chromatogram Trace.

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Figure 26. Mass Defect Filtered Chromatogram

The mass defect filtered chromatogram displays the sum of intensities of multiple centroid
masses within the specified mass defect range.

This table displays the parameters for calculating the mass defect filtered chromatogram trace.
Table 13. Mass Defect Filter Parameters (Sheet 1 of 2)
Parameter Description
Compound Name Specifies the name of the parent compound.
Compound Mass (amu) Specifies the exact mass or chemical formula in
atomic mass units (amu). You can also leave the
column empty for calculation.
• Format: 9999.9999
• Compound Mass: 0.0000 and/or empty.
When you add the chemical formula, the
Compound Mass box becomes non-editable.
Mass Defect (mmu) Displays the compound mass in milli mass units
(mmu), rounded to the third decimal place.
Use Chemical Formula Select to calculate the mass defect based on the
chemical formula.

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Table 13. Mass Defect Filter Parameters (Sheet 2 of 2)

Parameter Description
Chemical Formula Specifies the chemical formula used for calculation.
Species Specifies the element.
Charge Specifies the charge.
Low Mass (amu) The lower end of the mass defect range.
• When the compound mass is specified, the
value is relative to the compound mass.
• When the compound mass is empty or zero,
the value is an absolute value used for the mass
defect filter calculation.
High Mass (amu) The upper end of the mass defect range.
• When the compound mass is specified, the
value is relative to the compound mass.
• When the compound mass is empty or zero,
the value is an absolute value used for the mass
defect filter calculation.
Low Mass Defect (mmu) Three digits in mmu.
• When the compound mass is empty or zero,
the low mass defect value is an absolute value.
• When the compound mass is given, the value
is a relative value used for calculation.

Valid range: 0–500 mmu

Default: 50 mmu
High Mass Defect (mmu) Three digits in mmu.
• When the compound mass is empty or zero,
the High Mass Defect value is an absolute
value.
• When the compound mass is given, the value
is a relative value used for calculation.

Valid range: 0–500 mmu

Default: 50 mmu
Defect Range (amu) Low Mass Defect–High Mass Defect

Applying Mass Defect Filter in a Chromatogram

Y To apply a mass defect filter

1. Select the Chromatogram view, and then choose Chromatogram > Ranges in the
Workspace Options toolbar.

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2. In the Trace Type list, select Mass Defect Filter.

3. In the Mass Defect Range, click MDF Ranges.
4. In the Mass Defect Filter dialog box, enter the following parameters:
• Compound Name: Specify the compound name or leave the column empty.
• Compound Mass (amu): Specify the exact mass of the compound or leave the
column empty.
• Use Chemical Formula: Select if you want to use the chemical formula. The
compound mass and mass defect automatically populates when you use chemical
formula for calculation.
• Chemical Formula: Type the chemical formula that you want to use for calculation.
• Species: Select the species from the dropdown list.
• Charge: Select the charge from the dropdown list.
• Select the check box next to the first row.
• Enter the low mass, high mass, low mass defect, and high mass defect.
• Select the check box next to each row, and then repeat entering the multiple mass
range and mass range defect parameters to create a Multiple Mass Defect Filtered
trace.
5. Click Apply, and then click OK.
The mass defect filtered chromatogram displays.

Relative and Absolute Mass Range Calculation

Mass defect filtering of a chromatogram trace is calculated as either relative or absolute. When
you type a compound mass value in the Mass Defect Filter dialog box or use a chemical
formula, the mass defect range is calculated relative to the compound mass and its mass
defect. When you type zero or leave the compound mass column empty, the mass defect value
used for calculation is an absolute range based on the low and high mass, and the low and
high mass defect that you have entered.

Y To calculate the relative and absolute mass and mass defect values

1. In the Chromatogram Ranges view, select:

• Detector Type: MS
• Trace Type: Mass Defect Filter
2. In the Mass Defect Range column, click MDF Ranges.

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The Mass Defect Filter dialog box opens.

3. In the Compound Mass (amu) column, type the compound mass value for relative
calculation

–or–
Type zero or leave the column empty for absolute calculation.
When you type a compound mass value, the Mass Defect columns displays the first three
decimal values of the compound mass, rounded to the third decimal place. For example,
if you type 348.122, the application automatically populates the mass defect value as 122.
4. Select each row and type the low mass and high mass range values (for example, 300–305)
and the low mass and high mass defect values.
5. Click Apply, and then click OK.

This figure indicates how the relative and absolute calculation of mass and mass defect values
differ for different chromatogram traces in the FreeStyle application.
Figure 27. Relative and absolute mass defect and mass range changes for different
chromatograms

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Linking a Spectrum View to an MDF Chromatogram Trace

When you select an MDF chromatogram, the Spectrum view displays mass spectral peaks
corresponding to the mass defect filters applied to the chromatogram. When you select a
single scan or link a scan to an MDF chromatogram trace, the active or the linked spectrum
does the following:
• Displays the spectrum title as MMDF T: +c d Full ms2...
• Updates the filtered mass data corresponding to the active MDF chromatogram trace.
• Updates the Spectrum list with the filtered mass data corresponding to the active MDF
chromatogram trace.
• Makes the Write to .RAW feature available.
• Deactivates the NIST search and mzVault search options.
Figure 28. Spectrum linked to the MDF Chromatogram trace

Linked to MDF chromatogram trace

Y To link a spectrum view to an MDF chromatogram trace

1. In the Chromatogram Ranges view, select:

• Detector Type: MS.
• Trace Type: Mass Defect Filter.
2. In the Mass Defect Range column, click MDF Ranges.
The Mass Defect Filter dialog box opens.
3. In the Mass Defect Filter dialog box, enter the low mass, high mass range, low mass
defect, and high mass defect values.

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Setting up Instrument Status Traces

4. Click Apply, and then click OK.

The title of the Spectrum view describes the linked MDF chromatogram trace in the
following format:
MMDF T: +c d Full ms

Setting up Instrument Status Traces

You can specify traces for the MS detector’s status readbacks and compare these traces to
possible disturbances in the chromatographic data.

Y To add instrument status traces to the Chromatogram view

1. Select the Chromatogram view where you want to add the trace.
2. In the Workspace Options toolbar, choose Chromatogram > Ranges.
3. For each status readback of interest, do the following:
a. Add a plot to the Chromatogram view (see To add a chromatogram trace by using the
Chromatogram menu or To add chromatogram traces by using the Chromatogram
Ranges view).
b. In the Detector Type list of the Chromatogram Ranges view, select MS Trending.
c. In the Trace Type list, select a readback parameter.

This figure hows the ion transfer tube temperature readback as a function of time.
Figure 29. Status readback displayed in the Chromatogram view

Selected readback parameter Readback trace for the ion gauge

pressure during the 13 min run

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Displaying an EIC Trace

Displaying an EIC Trace

You can use the commands in the Tools area of the Spectrum – Workspace Processing toolbar
to define an EIC trace. The features in the Tools area are context-sensitive to whichever view
(Spectrum or Chromatogram) is active.

The Tools area has two icons for adding EIC traces to the Chromatogram view—the EIC
Masses icon, , and the EIC Range icon, . To add a plot for multiple discrete m/z
values, use the EIC Masses icon. To add a trace for contiguous m/z ranges, use the EIC Range
icon.

Tip To display an EIC trace for a single mass-to-charge value, double-click the
mass-to-charge label for the peak in the Spectrum view. The Spectrum view displays the
EIC trace for the selected mass-to-charge ratio ± 1 Da.

Y To display an EIC trace for multiple discrete m/z values

1. Select the Spectrum view.

2. In the Tools area of the Workspace Processing toolbar, click the EIC Masses icon, .
3. In the Spectrum view, click the mass-to-charge label for each peak that you want to use
for the EIC trace.
A red vertical marker indicates a selected peak. Figure 30 shows the selection of three mass
peaks.

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Figure 30. Spectrum view with the selection of three mass peaks

4. Click the EIC Masses icon, , again to create an EIC trace from the selected masses.
A new EIC trace appears at the bottom of the Chromatogram view (Figure 31), and the
selected masses appear in the Ranges column in the Chromatogram Ranges view.

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Figure 31. EIC trace created from three mass peaks

EIC trace

Y To specify the mass ranges for an EIC trace

1. In the Tools area of the Workspace Processing toolbar, click the EIC Range icon, .
2. In the Spectrum view, drag the pointer horizontally through a contiguous or
noncontiguous mass range to select the mass range that you want to use for the EIC trace.
A horizontal red line with start and end markers indicates the start and end of the selected
range (Figure 32).

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Figure 32. Two mass ranges selected in the Spectrum view

Mass range marker Mass range marker

3. To create an EIC trace from the selected mass ranges, click the EIC Range icon, ,
again.
The selected mass ranges appear in the Ranges column of the Chromatogram Ranges
view and a new EIC trace appears at the bottom of the Chromatogram view. Figure 33
shows the EIC trace for two noncontiguous mass ranges.

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Using the Scan Filters Page to Display a Filtered Chromatogram

Figure 33. EIC trace for two noncontiguous mass ranges

EIC
trace

Using the Scan Filters Page to Display a Filtered Chromatogram

Use the Scan Filters page or the Filters column of the Chromatogram Ranges view to display
filtered chromatograms.

Y To display a filtered chromatogram

1. Open the raw data file of interest.

2. In the Workspace Options toolbar, in the Filter area, click the Scan Filters icon, .
The Scan Filters page appears in the Info Bar. By default, the Time Range (min) box
contains an asterisk and the Track check box is clear. The asterisk means that the time
range is set to the full length of the acquisition time for the raw data file.

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3. (Optional) To limit the displayed time range to a portion of the acquisition time, do one
of the following:
• In the Time Range (min) box, type the retention-time range as follows:
Start time-End time
–or–
• Select the Track check box, and then in the Chromatogram view, drag the pointer
across the retention time of interest.
4. Select or type a filter in the filter list.
The filtered chromatogram appears in the Chromatogram view.

Setting Up the Display Options for a Chromatogram Trace

For the Chromatogram view, you can use the features in the Chromatogram – Display
Options toolbar to stack or overlay chromatogram traces, format and label the chromatogram
peaks and axes, specify how the application normalizes the chromatogram traces, and change
the y-axis title and scale.

Note Except for the Decimal Places icon, all the icons are available on the Chromatogram
– Display Options toolbar. The Decimal Places icon becomes available after you add the
Retention Time label to your chromatograms.

Although the Peak Area, Signal To Noise, and Height icons are always available, the
application adds these labels only to integrated peaks.

To customize the display options for chromatogram plots, see these topics:
• Formatting Chromatogram Plots
• Labeling Chromatogram Peaks or Local Maxima
• Normalizing Chromatogram Traces
• Changing the Y-Axis Title and Scale of the Chromatogram View

Formatting Chromatogram Plots

Use the Format area of the Chromatogram – Display Options toolbar to format the
chromatogram plots. You can display chromatogram traces as point-to-point graphs (line
graphs) or as discrete sticks for each scan. When the Chromatogram view contains multiple
plots, you can stack the plots or overlay them.

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With the default factory settings, the application displays multiple traces as stacked line
graphs, with the top graph plotted in black. The Stack, Overlay, Point-to-Point, and Stick
format options apply to all the plots. To select different colors for overlaid plots, select the
color for each active plot before you overlay them. Use the Elevation slider to change the
percentage overlay of the plots. Setting the slider full left overlays the traces to the same
elevation on the y axis. Use the Skew slider to offset the x axis of the plots. You cannot offset
the x axis when the traces are 100% overlaid to the same elevation on the y axis.

For information about all the display options, see Setting Up the Display Options for a
Chromatogram Trace.

Figure 34 shows two overlaid stick plots.

Figure 34. Overlaid stick plots

Labeling Chromatogram Peaks or Local Maxima

Use the Labels section of the Chromatogram – Display Options toolbar to label the
chromatogram traces. When using the default factory layout, the application labels the
retention time of each localized maxima in the chromatogram plots to two decimal places.
When you apply a peak detection algorithm or manually draw the chromatographic baseline,
the application only labels the retention time of the detected peaks.

For information about all the display options, see Setting Up the Display Options for a
Chromatogram Trace.

To label chromatogram traces, follow these procedures:

• To add the peak area, peak height, and signal-to-noise labels to each detected peak
• To remove a label
• To specify the format of the retention time label
• To change the number of decimal places for the numeric labels

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• To change the labeling threshold

• To display peak height or peak area in general format
• To display peak height and peak area in exponential format

Y To add the peak area, peak height, and signal-to-noise labels to each detected peak

1. Run a peak detection algorithm or manually draw the chromatographic baseline.

2. In the Labels area in the Chromatogram – Display Options toolbar, click the following
icons:
• To add the peak area label, click the Peak Area icon, .

Note The letters MA or AA next to the value indicate manual integration or

automatic integration, respectively.

• To add the peak height label, click the Peak Height icon, .

Note The letters MH or AH next to the value indicate manual integration or

automatic integration, respectively.

• To add the signal-to-noise label, click the Signal To Noise icon, .

Note The Avalon peak detection algorithm does not calculate a signal-to-noise
ratio. For the Genesis and ICIS peak detection algorithms, you can choose to
report the root mean square (RMS) noise values. For manual peaks, the
application calculates the RMS signal-to-noise ratio.

The letters SN next to the value indicate that it is a signal-to-noise ratio.

Y To remove a label

In the Labels area in the Chromatogram – Display Options toolbar, click the associated
icon.

Y To specify the format of the retention time label

Note By default, the Retention Time label is turned on and set to Decimal Minutes.

1. If the Retention Time label is turned off, click the Retention Time icon, , in the
Labels area in the Chromatogram – Display Options toolbar.

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2. Click the Retention Time Format icon, , and select the format for the RT label from
the dropdown list.

3. Click the Retention Time Decimal icon, , and select the number of decimal places
for the RT label from the dropdown list.

Y To change the number of decimal places for the numeric labels

Click Decimal Places and select a value from 0 to 5 from the list.

Y To change the labeling threshold

In the Label Threshold box, type a percentage from 0 to 100.

For a value of 0%, the application labels all the trace maxima or detected peaks. At a value
of 100%, the application labels only the highest data point on the trace. When the peak
apex of a detected peak falls below this height, the application does not label the peak.

Y To display peak height or peak area in general format

1. In the Labels area, click the Peak Height icon, , or the Peak Area icon, .
2. In the Labels Format area, click the General icon, .
The Chromatogram view displays the peak height and peak area in general format.

Peak Area and Peak Height

in general format

Y To display peak height and peak area in exponential format

1. In the Labels area, click the Peak Height icon, , or the Peak Area icon, .

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2. In the Labels Format area, click the Exponent icon, .

The Chromatogram view displays the peak height and peak area in exponential format.

Peak Area and Peak Height

in exponential (scientific notation) format

Filling the Chromatogram Peaks

Use the Peak Fill area in the Chromatogram – Displays Options toolbar to color the
chromatogram peaks. You can turn on or off the peak colors. The default color is blue.

Y To fill the chromatogram peaks

1. Click the Chromatogram view to make it active.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Peak Fill area, click the Color icon, .
The Color Palette dialog box opens where you can select a preset color or customize a
color.
4. Open the Peak Detection Info bar page.
5. Select the Peak Detection Algorithm, and then click Apply.
The peaks are filled with the selected color.
Figure 35. Peak Fill

6. To color the peaks of a PPD peak detection algorithm, select Apply to PPD Peaks.
7. To turn off the color, in the Peak Fill area, click the Fill icon, .

Note The Fill icon displays or hides the color of the peaks.

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Normalizing Chromatogram Traces

Use the Normalization area of the Chromatogram – Display Options toolbar to normalize
chromatogram traces. You can apply local or global normalization or turn off normalization.

Changing the normalization type changes the y-axis scaling:

• Local normalization scales each trace independently to its largest peak in the displayed
time range.
• Global normalization scales each trace to the largest peak across the traces in the displayed
time range.
• Turning off normalization scales each trace to the largest peak across the traces in the full
time range, regardless of the displayed time range.

Follow the procedures in these topics:

• Applying Local Normalization
• Applying Global Normalization
• Turning Off Normalization
• Changing the Y-Axis Range for Normalized Chromatograms

For information about all the display options, see Setting Up the Display Options for a
Chromatogram Trace.

Applying Local Normalization

When you apply local normalization (the default setting), the application normalizes the
chromatogram traces so that, within the displayed time range, the most intense peak for each
trace is set to 100 percent of the y-axis scale.

Y To apply local normalization

1. Click the Chromatogram view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Normalization area, click the Local icon, .

Figure 36 shows an example of local normalization. Each trace is independently scaled to

its largest peak in the displayed time range, which is the full time range.

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Figure 36. Local normalization with full time range displayed

Figure 37 shows the TIC traces for the same scan filters as those shown in Figure 36, but
only the 1 to 2 min time range is displayed. In this case, each trace is independently scaled
to its largest peak in the displayed time range of 1 to 2 min.
Figure 37. Local normalization for the 1 to 2 minute time range

Applying Global Normalization

For global normalization, the application normalizes the chromatogram traces so that the
most intense peak of all traces is scaled to 100% of the y axis.

Y To apply global normalization

1. Click the Chromatogram view to select it.

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2. Click the Display Options tab to open the Display Options toolbar.
3. In the Normalization area, click the Global icon, .
Figure 38 shows an example of global normalization. Both traces are scaled to the largest
peak across the traces, rather than being independently scaled to the largest peak in each
trace.
Figure 38. Global normalization with the full time range displayed

Figure 39 shows the TIC traces for the same scan filters as those shown in Figure 38, but
only the 1 to 2 minute time range is displayed. In this case, each trace is scaled to the
largest peak across the traces in the displayed time range of 1 to 2 minutes.
Figure 39. Global normalization with the 1 to 2 minute time range displayed

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Setting Up the Display Options for a Chromatogram Trace

Turning Off Normalization

When you turn off normalization, the application scales all the traces by using the maximum
and minimum absolute values in the full time range, regardless of the time range displayed.

Y To turn off normalization

1. Click the Chromatogram view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Normalization area, click the Off icon, .
A comparison of Figure 40 and Figure 41 shows the effect of turning off normalization.
Regardless of the time range displayed, the traces are scaled to the largest peak across the
traces in the full time range. In addition, the Max box displays the absolute intensity of
the largest peak (apex height).
Figure 40. Normalization turned off with the full time range displayed

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Figure 41. Normalization turned off with the 1 to 2 minute time range displayed

Changing the Y-Axis Range for Normalized Chromatograms

Use the Min and Max boxes in the Normalization area of the Chromatogram – Display
Options toolbar to change the y-axis range from its default of full zoom (0–100%) to another
zoom level.

Note Regardless of whether the y-axis scaling (Y Scale) is set to Absolute or % Relative,
when normalization is turned on (local or global), the Min and Max boxes accept positive
and negative values as percentages of the full y-axis scale. The Max value must be greater
than the Min value.

For example, to zoom in on the –10 to 50% portion of the y axis, type –10 in the Min. box
and 50 in the Max. box.

Figure 42 shows the result.

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Figure 42. Modified y-axis range of –10 to 50%

Changing the Y-Axis Title and Scale of the Chromatogram View

Use the Y Axis and Y-Scale area of the Chromatogram – Display Options toolbar to change
the y-axis title and scale of the Chromatogram view.

Follow the procedures in these topics:

• Changing the Y-Axis Title
• Changing the Y-Axis Scale

Changing the Y-Axis Title

Makes available the Separate Axis icon in the y-axis area to change the y-axis title of the
individual traces of the Chromatogram view.

Y To change the Y-Axis title of the Chromatogram view

1. Click the Chromatogram trace to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Y Axis area, click the Separate Axis icon, .
A text box opens where you can type a y-axis title.
4. (Optional) type a new y-axis title and press RETURN.
The new title appears in the active Chromatogram trace in the Chromatogram view.

Note When you add a new Chromatogram trace, the y-axis title of the active
Chromatogram trace reflects on the new trace.

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Changing the Zoom Level of a Chromatogram

Changing the Y-Axis Scale

Relative abundance is the default y-axis scale (units) for the Chromatogram view. You can
change the y-axis units of individual traces of the Chromatogram view by enabling the
Separate Axis option in the Y Axis area. Use the Default Workspace Options to change the
y-scale units for each detector type. For information on how to set the units according to the
detector type, see Workspace Options Page.

Y To change the Y-Axis units from relative abundance to absolute

1. Click the trace in the Chromatogram view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Y-Scale area, click |Y|Absolute.
4. You can view the y-axis units in either exponent (10×) or general form by clicking or
in the Y-Scale area.
Figure 43 shows a Chromatogram view with the y-axis scale set to absolute (where the
title is intensity) and normalization set to local.
Figure 43. Units for the y-axis set to absolute intensity

Changing the Zoom Level of a Chromatogram

You can zoom in on or out of a region of a chromatogram by using the Zoom Options toolbar
or the mouse pointer.
Follow these procedures:
• To incrementally zoom in and out on the x axis
• To zoom in on a specific section of the x axis
• To incrementally zoom in and out on the y axis
• To reset the zoom level of the x and y axes

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Y To incrementally zoom in and out on the x axis

1. Select the view.

2. To zoom in, in the Zoom Options toolbar, click the Zoom In X icon, .
Each click zooms in on a 50% smaller section of the x axis—that is, it decreases the
displayed range by a factor of 2.
3. To zoom out, in the Zoom Options toolbar, click the Zoom Out X icon, .
Each click increases the displayed time range by 200%.

Y To zoom in on a specific section of the x axis

Drag the pointer horizontally across the specific section of the x axis.

Y To incrementally zoom in and out on the y axis

1. Select the view.

2. In the Zoom Options toolbar, click the Zoom In Y icon, .
Each click zooms in on a 50% smaller section of the y axis. For example, in a
Chromatogram view where the y axis is set to relative abundance, clicking once zooms in
on the 0 to 50% range, clicking twice zooms in on the 0 to 25% range, and so on.
3. To incrementally undo the zoom level, do the following:
• Click the Zoom Out Y icon, .
–or–
• Right-click the view and choose Undo Zoom.
Each click zooms out to a 200% larger section of the y axis.

Y To reset the zoom level of the x and y axes

1. Select the view.

2. Do any of the following:
• In the Zoom Options toolbar, click the Reset icon, .
• Right-click the view and choose Reset Scaling.

Automatically Detecting and Integrating Chromatographic Peaks

Use the Peak Detection page in the Info Bar to select and apply any of the automated peak
detection algorithms—Genesis, ICIS, Avalon, or PPD. When using the Genesis or ICIS
algorithm, you can manually select the chromatographic time range that the application uses
to determine the baseline noise level.

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Follow these procedures:

• To display the Peak Detection page
• To define the identification criteria for a component
• To run any of the peak detection algorithms
• To run the elemental composition algorithm
• To reset the zoom level in the Chromatogram view

Y To display the Peak Detection page

1. Open a raw data file (.raw) or a sequence file (.sld).

The Peak Detection page appears in the Info Bar.
2. Click the Peak Detection tab.

Y To define the identification criteria for a component

1. In the Identification area, select one of the following from the Returned Peaks dropdown
list:
• All Peaks: Select to identify all peaks in the chromatogram based on the selected
algorithm for component identification.
• Nearest Peak: Select to identify the nearest peak in the chromatogram within the
specified time window.
• Highest Peak: Select to identify the highest peak in the chromatogram within the
specified time window.
2. When Nearest Peak or Highest Peak is selected, in the Expected Time (min) box, type or
select the expected retention time for the component. The valid range is 0 to 999
minutes.
3. When Nearest Peak or Highest Peak is selected, in the Window (sec) box, type or select
the retention time window for the component. The valid range is 1.0 to 999.0 seconds.
4. Specify the component name used in the processing method.

Y To run any of the peak detection algorithms

1. In the list, select one of these algorithms:

• Genesis—Use for mass spectrometry data.
• ICIS—Use for mass spectrometry data.
• Avalon—Use for UV or PDA data. This algorithm detects negative chromatographic
peaks more accurately than the ICIS or Genesis algorithms.
• PPD—Use to minimize the number of optimization parameters.

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2. When the Chromatogram view contains multiple plots, do one of the following:
• To process all the plots, select the Apply to All Plots check box.
• To process only the selected plot, clear the Apply to All Plots check box.
3. When necessary, modify the parameters settings for the selected algorithm.
For information about the parameters settings for each algorithm, see these topics:
• Genesis Peak Detection Page
• ICIS Peak Detection Page
• Avalon Peak Detection Page
• PPD Peak Detection Page
For information about selecting the manual noise region for the ICIS or Genesis
algorithms, see Selecting the Manual Noise Region for the Genesis and ICIS Algorithms.
4. (Optional) To save the modified settings as the default settings for the selected algorithm,
click Save As Defaults.

Note You can view the saved settings on the Peak Detection page of the Default
Options Configuration dialog box. See Appendix A, “FreeStyle Default Settings.”

5. Depending on the algorithm, do one of the following:

• For the Genesis, ICIS, or PPD algorithm, click Apply.
• For the Avalon algorithm, click Auto Calc Initial Events. When necessary, customize
the integration by adding more events to the table. Each time you add an event, the
algorithm automatically reintegrates the plots.
After the peak detection finishes, the application does the following:
• Adds the detected peaks to the peak list in the Peaks List view.
• Colors the integrated region of the detected peaks in the Chromatogram view.
• Draws a baseline under the integration region.
• Adds square integration markers at the beginning and end of the integration region.
You can change the integration region by dragging the markers.

Note When the instrument method used to acquire the raw data file specifies the
compound names of the analytes, the application displays these names above the
detected peaks. The instrument control software for the Thermo Scientific TSQ
Endura™ and TSQ Quantiva™ mass spectrometers provides this feature.

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Y To check the integration for each chromatographic peak

1. If you applied the algorithm to multiple plots, in the Peaks List view, select the plot from
the trace list.

Note The Peaks List view defaults to displaying a list of all the peaks (All Peaks)
detected across the chromatogram plots.

Figure 44 shows the second trace (Trace 2) selected from a list of three chromatogram
plots.
Figure 44. Peak list for Trace 2

2. To check the integration of a peak, select its row in the peak list.
In the Chromatogram view, the application zooms in on the selected peak.
Figure 45 shows the selection of the second peak in the second chromatogram plot.

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Selecting the Manual Noise Region for the Genesis and ICIS Algorithms

Figure 45. Zoom level set to the second peak in the second chromatogram plot

Y To run the elemental composition algorithm

Select the Empirical Formula check box.

The Empirical Formula column appears to the left of the File Name column.

Y To reset the zoom level in the Chromatogram view

In the Peaks List view, click the Reset icon, .

Selecting the Manual Noise Region for the Genesis and ICIS
Algorithms
You can manually select the noise region for the Genesis and ICIS peak detection algorithms.

Y To specify the manual noise region for the Genesis or ICIS peak detection algorithm

1. Open the Peak Detection page (see To display the Peak Detection page).
2. From the peak detection algorithm list, select ICIS or Genesis, and then specify the
appropriate parameter settings.
3. Click the Workspace Processing tab to display the Workspace Processing toolbar.
4. In the Peak Detection area of the toolbar, click Select Manual Noise Range.
5. Drag the pointer horizontally across a region of the chromatographic baseline (the x axis).

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Manually Adding, Undoing, and Deleting Chromatographic Peaks

In the Chromatogram view, the application indicates the noise region with a red line and
updates the peak integration. On the Peak Detection page, it automatically selects the
Manual Noise Region check box and populates the RT Range box with the selected time
range. In the Peaks List view, the application updates the detected peaks list.
6. Do the following:
• To change the noise region, in the Workspace Processing toolbar, click Select Manual
Noise Range, and then drag the pointer horizontally across a different region of the
chromatographic baseline.
• To undo the manual noise region, in the Workspace Processing toolbar, click Clear
Manual Noise Range, and then drag the pointer horizontally across the highlighted
noise region.
The application automatically updates the peak detection and integration.

Manually Adding, Undoing, and Deleting Chromatographic Peaks

You can manually add peaks to a chromatogram and the Peaks List view without running a
peak detection algorithm. You can also delete peaks from the Peaks List view or undo the last
peak that you added manually.

Follow the procedures in these topics:

• Manually Adding Chromatographic Peaks
• Undoing the Last Manually Added Peak
• Deleting Chromatographic Peaks

Manually Adding Chromatographic Peaks

Y To manually add peaks to the peak list

1. Click a Chromatogram view to select it.

2. Turn on the Add Peak pointer, click Add Peak in the Chromatogram – Workspace
Processing toolbar.
3. Drag the pointer, , along the bases of the peaks that you want to integrate and add
to the peak list.
After the peak detection finishes, the application does the following:
• Adds the detected peaks to the peak list in the Peaks List view, and identifies their
integration method as Manual.
• Colors the integrated region of the detected peaks blue.
• Draws a baseline under the integration region.

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• Adds square integration markers at the beginning and end of the integration region.
You can change the integration region by dragging the markers.
Figure 46 shows a manual peak and its integration markers.
Figure 46. Manual peak with integration markers and peaks list with a manual peak
Integration
markers

Start End

Manual
peak

4. To turn off the Add Peak pointer, click the Add Peak icon again.

Undoing the Last Manually Added Peak

Y To undo the last peak that you added manually

Right-click the chromatogram and choose Undo Add Peak.

Deleting Chromatographic Peaks

You can delete chromatographic peaks, regardless of their integration method.

Y To delete chromatographic peaks

1. Click the Chromatogram view to select it.

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Importing Components from a Processing Method

2. To turn on the Delete Peak pointer, click Delete Peak in the Chromatogram –
Workspace Processing Toolbar.
3. Click the peak that you want to delete.

Delete Peak
pointer

4. To turn off the Delete Peak pointer, click the Delete Peak icon again.

Importing Components from a Processing Method

Use the Import Component feature to import components from a processing method to the
existing workspace. The imported processing method contains component identification
parameters that you can apply to the current or new traces in the chromatogram. Processing
methods are PMD files.

Y To import components

1. Click the Chromatogram view to make it active.

2. In the Workspace Processing toolbar, click Import Component.
The Import Component from Processing Method dialog box opens.
Figure 47. Import Component from Processing Method dialog box

3. In the Select processing method file box, click the Browse icon to locate the .pmd file.
4. In the Open File dialog box, select the .pmd file, and then click Open.
The application imports the components and automatically displays the first component.
5. To change the component, click the Select component down arrow icon, and then select
another component.
6. Select the Current Trace or New Trace option to add the component to the current or
new trace.
7. Click Apply, and then OK.

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Exporting Scans for a Filtered Chromatogram to a New Raw Data File

The peak detection is applied to the current or new trace based on the selected option.
The component parameters are imported from the processing method to the Peak
Detection Info bar page and the Chromatogram Ranges.
Note The Component Name box of the Peak Detection Info page displays the imported
component, and you can edit the component name.

Exporting Scans for a Filtered Chromatogram to a New Raw Data File

Raw data files can contain gigabytes of data. To work with only the scans related to the
displayed time range of a filtered chromatogram, you can export the scans to a new raw data
file, which will have a smaller file size. The new raw data file does not include the instrument
method or sample information.

Y To export a scan for a filtered chromatogram to a new raw data file

1. Select the chromatogram trace.

2. In the Workspace Options toolbar, click Exports and choose Write to .RAW from the
dropdown menu.

Note When the raw data file contains Advanced Peak Determination (APD) data—
such as species ID, monoisotopic mass, and top peak in the cluster—the exported file
does not include the APD data.

In Figure 48, the scan header for the selected trace indicates that it is a filtered trace for
the full MS scans (F: MS).
Figure 48. Chromatogram view with filtered traces

Filtered
trace

The Export Data dialog box opens to the location of the original raw data file. By default,
the application appends _FS to the file name of the original raw data file.

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3. Click Save.
A progress bar indicates the number of scans written to the new raw data file.

Note When the chromatogram trace is filtered, the application renumbers the scans
from 1 to the number written to the new raw data file.

The application records the creation of new raw data file in the Audit Trail.

Working with Sequences

A sequence is a list of raw data files where the file names link to the actual raw data files. You
can open an existing sequence or create a new sequence from a set of raw data files.

To open an existing sequence, see Opening Raw Data Files or Sequence Files.

Follow the procedures in these topics:

• Creating a New Sequence
• Comparing Chromatogram Traces in a Sequence

Creating a New Sequence

You can create a sequence for any raw data file set.

Y To create a new sequence

1. Do one of the following:

• Choose File > Create Sequence.
–or–
• In the Workspace Options toolbar, click Create Sequence.
2. Browse to and select a set of raw data files.
3. Click Open.
The Sequence File page opens in the Info bar. By default, the new sequence list is named
NoName.sld.
4. To save the sequence, do the following:
a. At the bottom of the Sequence File page, click Save Sequence.
b. Browse to an appropriate file directory, name the file, and click Save.

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Comparing Chromatogram Traces in a Sequence

To compare one or more fixed traces against other traces, specify the fixed traces as reference
traces in the Chromatogram Ranges view.

Y To compare chromatogram traces in a sequence

1. Open a sequence (see Using Workspace Pages) or create a sequence (see Working with
Sequences).
2. In the sequence list, select the raw data file that you want to use as a reference file.
The Chromatogram view displays the TIC trace for the raw data file.
3. Specify the reference trace as follows:
a. In the Workspace Options toolbar, choose Chromatogram > Ranges to open the
Chromatogram Ranges view.
b. Define the trace that you want to use as a reference trace. For example, select the
detector type, trace type, filter, and so on.
c. Copy this definition to the last row in the Chromatogram Ranges view by selecting
its corresponding check box in the Display column.
A copy of the first chromatogram trace appears below it.
d. Select the check box in the Reference column of the row corresponding to the
reference trace.
4. Click the raw data files in the sequence to compare their chromatogram traces to the
reference chromatogram trace.
Figure 49 shows a Chromatogram view with two plots. The comparison trace for the
selected file is on the top, and the reference trace is on the bottom.

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Figure 49. Chromatogram view with a comparison trace and a reference trace

Comparison trace

Reference trace

Selected comparison file Reference trace definition

Chromatogram-specific Toolbars
Use the following toolbars to format the Chromatogram view, detect chromatographic peaks,
and work with sequence lists.
• Chromatogram – Display Options Toolbar
• Chromatogram – Workspace Processing Toolbar
• Sequence Toolbar

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Chromatogram – Display Options Toolbar

Use the features on the Chromatogram – Display Options toolbar to customize the
Chromatogram view.

Y To display the Chromatogram – Display Options toolbar

1. Click a Chromatogram view to select it.

2. Click the Display Options tab to open the Display Options toolbar.

Figure 50 shows the Chromatogram – Display Options toolbar, and Table 14 describes the
toolbar features.
Figure 50. Chromatogram Display Options toolbar

Table 14. Chromatogram – Display Options toolbar (Sheet 1 of 4)

Command Description
Format area

Use the features in the Format area to modify the appearance of the Chromatogram view.
Plot Options menu
Stack Vertically stacks the chromatogram traces.
Overlay Vertically overlays the chromatogram traces with optional horizontal skew (time
offset).
Sets the skew angle (time offset) to a value from 0–45 degrees for an overlay
arrangement of chromatogram traces.

To set the skew, drag the Skew slider.

Sets the vertical spacing for an overlay arrangement of chromatogram traces.

To set the vertical spacing, drag the Elevation slider. Move the Elevation slider to
the farthest left to overlay the plots on top of each other.
Plot Style menu
Point-to-point Connects the signal data points to form a continuous curve.
Stick Displays the signal data points by using vertical lines.

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Table 14. Chromatogram – Display Options toolbar (Sheet 2 of 4)

Command Description
Displays the color palette, where you select the colors of the chromatogram traces.

Labels area

Use the features in the Labels area to annotate chromatogram peaks.

Retention Time Adds a retention time label (in minutes) above each local maxima or
chromatogram peak.

The order of chromatogram labels for an undetected peak, from top to bottom, is
retention time, base peak, and scan number. The Chromatogram view displays
the retention time on all peaks that meet the selection criteria.

The letters RT to the left of the value indicate a retention time.

Retention Time Format Specifies the units and format for the retention time label.

Selections: Decimal Minutes, Decimal Minutes with Units, Minutes and Seconds,
Seconds, Seconds with Units
Decimal Places Changes the number of decimal places in the retention time label.

Range: 0–5
Peak Area Adds a peak area label above each detected chromatogram peak.

(of integrated peaks) The letters MA or AA next to the value indicate manual integration or automatic
integration, respectively.
Base Peak Adds a base peak mass-to-charge ratio label (in m/z) above each local maxima or
each detected chromatogram peak.

The letters BP next to the value indicate a base peak.

Signal To Noise Adds a signal-to-noise ratio label above each detected chromatogram peak.

(of integrated peaks) The Avalon peak detection algorithm does not calculate a signal-to-noise ratio.
For the Genesis and ICIS peak detection algorithms, you can choose to report the
root mean square (RMS) noise values. The application calculates the RMS
signal-to-noise ratio for manual peaks.

The letters SN next to the value indicate that it is a signal-to-noise ratio.

Scan Number Adds the active scan number label above the chromatogram peak.

The letter S# next to the value indicates the scan number.

Height Adds a peak height above each detected chromatogram peak.

(of integrated peaks) The letters MH or AH next to the value indicate manual integration or automatic
integration, respectively.

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Table 14. Chromatogram – Display Options toolbar (Sheet 3 of 4)

Command Description
Label Threshold Sets the percentage of highest data point in the trace to 100%.

The application labels only the local maxima or chromatographic peak apexes that
are above the specified height threshold.

Range: 0–100; default: 0%

Labels Format area
Exponent Sets the label unit in exponent form (scientific notation) for Peak Area and
Height.
General (Default) Sets the label unit in general form for Peak Area and Height.
Peak Fill area

Use the features in the Peak Fill area to color the chromatogram traces. See Filling the Chromatogram Peaks.
Fill Turns on or off peak fill in the active chromatogram view.

Color Opens the color palette, where you can select the colors to fill the integrated
peaks.
Apply to PPD peaks Fills the PPD peaks in the active chromatogram view with the selected color from
the color palette.
Normalization area

Use the features in the Normalization area to specify how the application normalizes the chromatogram traces. See
Normalizing Chromatogram Traces.
Local Normalizes each chromatogram trace with respect to the intensity of the most
intense peak in that trace.
Global Normalizes the chromatogram traces so that the most intense peak of all
chromatograms is 100 percent.
Off Scales all chromatograms by using the maximum and minimum absolute values.
Note When you set the normalization to Off, the y-axis scale to Absolute, and
change the trace type, the new trace might not display. To remedy this, set the
normalization to Local and then to Off.
Min. Displays the minimum of the y axis. Enter a value in the box to change the
minimum. The application indicates whether the value is a percentage or an
intensity.
Max. Displays the maximum of the y axis. Enter a value in the box to change the
maximum value. The application indicates whether the value is a percentage or an
intensity.

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Table 14. Chromatogram – Display Options toolbar (Sheet 4 of 4)

Command Description
Y Axis area

Use the features in the Y Axis area to specify how the application labels and displays the y axis. See Changing the
Y-Axis Title and Scale of the Chromatogram View.
Show Labels Shows or hides the y-axis label.
Offset Axis Sets the location of the displayed plot at a specified distance from the y axis.

The y-axis offset moves the x axis slightly to the right of the y axis so that you can
see the plot details at low x-axis values.
Separate Axis Sets the Separate Axis title for the active Chromatogram trace.
Note Makes the Separate Axis icon available in the text box. Use this box to
change the y-axis title.
Y-Scale

Use the features in the Y-Scale area to specify how the application scales the y axis. See Changing the Y-Axis Title and
Scale of the Chromatogram View.
Relative Scales the chromatograms traces by using percentages.

Absolute Scales the chromatograms traces by using the absolute values.

Note When you set the normalization to Off, the y axis to Absolute, and
change the trace type, the new trace might not display. To remedy this, set the
normalization to Local and then to Off.
Exponent Sets the Y-Scale unit in exponent form.
General Sets the Y-Scale unit in general form.
X Axis

Use the features in the X Axis area to specify how the application labels and displays the x axis.
Show Labels Shows or hides the x-axis label.
Offset Axis Sets the location of the displayed plot at a specified distance from the x axis.

The x-axis offset moves the y axis up slightly so that you can see the plot details at
low y-axis values.
Legend
Trace Title Moves the title of the chromatogram trace from above the trace to beside it.

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Chromatogram – Workspace Processing Toolbar

Use the features in the Peak Detection area of the Workspace Processing toolbar to detect
peaks in chromatogram traces.

Y To display the Chromatogram Workspace Processing toolbar

1. Click a Chromatogram view to select it.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.

The Peak Detection Area and the Tools Area are unique to the Chromatogram view.

Peak Detection Area

Figure 51 shows the Peak Detection area of the Workspace Processing toolbar. The Delete
Peaks menu becomes available after you apply a peak detection algorithm or manually add a
peak.
Figure 51. Peak Detection area of the Workspace Processing toolbar

Table 15 describes the Peak Detection area features.

Table 15. Workspace Processing toolbar – Peak Detection area commands (Sheet 1 of 2)
Command Description
Detect Peaks menu
Detect in Active Plot Runs the Genesis, ICIS, or Avalon peak detection algorithm
on the active chromatogram plot.

Click a chromatogram plot to make it active. The

Chromatogram view shades the active plot area gray. See
Automatically Detecting and Integrating Chromatographic
Peaks.
Detect in All Plots Runs the Genesis, ICIS, or Avalon peak detection algorithm
on all chromatogram plots. See Automatically Detecting and
Integrating Chromatographic Peaks.
Add/Delete Peak menu
Add Peak Adds a peak to the peak list. See Manually Adding
Chromatographic Peaks.

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Table 15. Workspace Processing toolbar – Peak Detection area commands (Sheet 2 of 2)
Command Description
Delete Peak Deletes a peak from the peak list. See Deleting
Chromatographic Peaks.

Available after you apply a peak detection algorithm or

manually add a chromatographic peak.
Peaks List Displays the Peaks List view. When you run a peak detection
algorithm, the Peaks List view lists the peaks that the
application found in the chromatogram. You can also
manually add peaks to the peak list with the Add Peak icon.
Manual Noise Range menu
Select Manual Noise Freezes the Chromatogram view (temporarily turns off
Range zooming with the mouse pointer) so that you can use the
mouse pointer to define a noise region (see Selecting the
Manual Noise Region for the Genesis and ICIS Algorithms).
Clear Manual Noise Freezes the Chromatogram view (temporarily turns off
Range zooming with the mouse pointer) so that you can use the
mouse pointer to clear the selection of a manual noise region
(see Selecting the Manual Noise Region for the Genesis and
ICIS Algorithms).

Tools Area
Figure 52 shows the Tools area of the Workspace Processing toolbar.
Figure 52. Tools area of the Workspace Processing toolbar

Table 16. Workspace Processing toolbar – Tools area commands (Sheet 1 of 2)

Command Description
Average Spectrum Averages the mass spectra over a selected retention-time range.
Clicking the icon toggles between Average Range and Zoom
Range methods. A blue ball, , indicates the active
method.

To create an average spectrum, see Averaging Spectra.

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Table 16. Workspace Processing toolbar – Tools area commands (Sheet 2 of 2)

Command Description
Avg Range Drag the cursor across the x-axis to choose an RT range,
average the scans, and update the active spectrum.
Note Click in the box to activate the Average Range
method.
Zoom Range Enter a retention time (RT) range within the chromatogram
RT limits and then press ENTER. The view rescales to
display the chromatogram within the entered time range.
Note Click in the box to activate the Zoom Range method.
Subtracts the mass spectrum of one retention-time range from
the active mass spectrum, as entered in the Range1 box.

To create a background subtracted spectrum, see Subtracting

Background Spectra.
Subtracts the mass spectra of two retention-time ranges from
the active mass spectrum, as entered in the Range1 and
Range2 boxes.

To create a background subtracted spectrum, see Subtracting

Background Spectra.

Sequence Toolbar
Use the Sequence toolbar features to determine which traces appear in the Chromatogram
view as you click through the raw data files in a sequence.

Y To display the Sequence toolbar

1. Open a sequence (see Using Workspace Pages) or create one (see Working with
Sequences).
2. If the Sequence File page is not open, click the Sequence File tab in the Info Bar.

Tip The Sequence toolbar is visible only when the Sequence File page is open.

Figure 53 shows the Sequence toolbar, and Table 17 describes the toolbar icons.
Figure 53. Sequence toolbar

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Table 17. Sequence toolbar icons

Icon Description
Replace All Traces Replaces all the raw data files in the chromatogram ranges list—
and their chromatogram traces in the Chromatogram view—with
the raw data file that you select in the sequence.

Select Replace All Traces, and then select a raw data file in the
sequence on the Sequence File page for replacement.
Note The application does not replace a trace that is set as a
reference trace (its Reference check box is selected) with the
selection from the sequence.
Current Trace Replaces only the raw data file that you select in the
chromatogram ranges list—and its chromatogram trace displayed
in the Chromatogram view—with the raw data file that you select
in the sequence.

Select a trace row in the chromatogram ranges list, select Current

Traces, and then select a raw data file in the sequence for
replacement.
Note The application does not replace a trace that is set as a
reference trace (the box in the Reference column is selected) with
the selection from the sequence.
New Trace Adds new raw data files to the chromatogram ranges list and
displays their chromatogram traces in the Chromatogram view.

Select New Trace, and then select one or more raw data files in the
sequence to add to the list.

Chromatogram-Specific Views
For information about the chromatogram-related views, see these topics:
• Chromatogram View
• Chromatogram Ranges View

Chromatogram View
Use the Chromatogram view to display chromatogram traces. You can delete a
Chromatogram view, add multiple chromatogram traces to each Chromatogram view, and
add multiple Chromatogram views to the Workspace.

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Figure 54 shows an example of a Chromatogram view with two traces. For information about
the scan header that appears at the top right of each chromatogram trace, see Scan Headers
and Scan Header Abbreviations.
Figure 54. Chromatogram view with two traces
Chromatogram view Total data
number acquisition time Normalization
level

Trace type,
detector type,
and file name

Delete icon

Normalization level,
trace type, detector type,
scan filter, and file name

Note You can set a minimum trace height value, in centimeters, in the Default Workspace
Options page. When you adjust the height of a Chromatogram view, if the height of the
traces becomes smaller than the set minimum value, a scrollbar automatically appears to
the right of the view.

Right-clicking a Chromatogram view displays a shortcut menu (Figure 55) with the
commands described in Table 18.
Figure 55. Shortcut menu for a Chromatogram view

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Table 18. Chromatogram view shortcut menu commands

Command Description
Reset Scaling Resets the scaling of all the plots in the Chromatogram
view.
Copy To Clipboard Copies an image of the Chromatogram view to the
Clipboard.
Undo Zoom Undoes the last zoom.
Redo Zoom Reapplies the last zoom that you undid.
Ranges Adds the Chromatogram Ranges view to the workspace
display.
Insert Chromatogram Inserts a copy of the selected chromatogram trace.
Undo Delete Chromatogram Displays the most recently deleted chromatogram trace.
Undo Manual Integration Removes the most recently added manual peak from the
Chromatogram view and the Peaks List view.
Average Spectrum Lets you create an average spectrum by dragging the
cursor across the x-axis to choose an RT range.

These toolbars are available when a Chromatogram view is selected:

• Chromatogram – Workspace Processing Toolbar
• Chromatogram – Display Options Toolbar
• Zoom Options Toolbar
• Text and Graphic Annotation Toolbar

Related Topics
• Reviewing Chromatographic Data

Chromatogram Ranges View

Use the Chromatogram Ranges view (Figure 56)to select which chromatogram traces to
display in a Chromatogram view. For instructions, see Defining a Chromatogram Trace from
the Chromatogram Ranges View.

Note For information about exporting the tabular data in the Chromatogram Ranges
view to a CSV file, see Exporting or Printing the Contents of a View or Workspace. You
can paste the contents of a CSV file to the Chromatogram Ranges view if the table
columns match.

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Y To open the Chromatogram Ranges view

In the Workspace Options toolbar, choose Chromatogram > Ranges.

To add, delete, and move rows in the Chromatogram Ranges view, see these topics:
• Using the Fill Down Feature
• Moving a Row
• Deleting Rows

Figure 56 shows an example of the Chromatogram Ranges view.

Figure 56. Chromatogram Ranges view showing the entry of a chemical formula

Field Chooser icon

Fill Down icon

Table 19 describes the parameters and icons for the Chromatogram Ranges view. The
following columns have corresponding pages in the Info Bar: Detector Type, Trace Type, and
Filter.
Table 19. Chromatogram Ranges view parameters and icons (Sheet 1 of 4)
Parameter/Icon Description
Display Select this check box to display the chromatogram trace in a Chromatogram view.
Clear this check box to remove the trace.
File Name Displays the path and name of the raw data file.

To display the Open Raw File dialog box, click at the end of the file name.
Detector Type Select the detector type. The application autosenses the available detector types based
on the information in the raw data file. For details, see Detector Type Page.
Filter Select or type a filter to apply to the raw data file. The application autosenses the
metafilters (including SRM or compound filters) in the raw data file in this order: MS,
MS2, ETD, HCD, and then the individual scan filter list. For data files with more than
one MS/MS order, enter MSn to include all the MS/MS data.

For grouped filters, the Filter dropdown list displays only the first filter in the group.
The Spectrum view displays the actual filter for the scan. For more information, see
Scan Filter Parameters and Scan Filters Page.

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Table 19. Chromatogram Ranges view parameters and icons (Sheet 2 of 4)

Parameter/Icon Description
Trace Type Select the trace type as follows:
• For MS, select TIC, Base Peak, Mass Range, Neutral Fragment, or Mass Defect
Filter.
• For PDA, select Total Scan, Wavelength Range, or Maximum Spectrum.
• For MS Trending, select one of the instrument status parameters, such as the API
source voltage.
Mass Defect Range When the Trace Type is set to Mass Defect Filter, this icon opens the Mass Defect Filter
dialog box.
Ranges Specify the m/z ranges of the displayed spectrum, such as 443.2, 534.6, 600–800. The
application autosenses the default range from the raw data file. If the field is inactive, it
is not applicable based on the trace type.
Smoothing Select which type of smoothing, if any, to apply to the chromatogram trace.
• Gaussian: Uses weighting coefficients corresponding to a Gaussian peak shape to
average each data point with neighboring points to give the displayed value.
• Moving mean: Uses equal weighting to average each data point with neighboring
points to give the displayed value.
Chemical Formula Clicking this table cell (not the header) opens the following dialog box.

Select Chemical Formula or Peptide:

• For a compound, select Chemical Formula, use the symbols in the periodic table
to specify the elements in the chemical formula, select the ionization species, and
type or select the charge of the adduct ion of interest.
Example: For calcium copper titanate (which is made up of one calcium atom,
three copper atoms, four titanium atoms, and twelve oxygen atoms), enter the
chemical formula: CaCu3Ti4O12.
Note To create a custom adduct, select Chemical Formula from the dropdown list,
enter any valid chemical formula, select a species, and type or select the charge of the
adduct ion.

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Table 19. Chromatogram Ranges view parameters and icons (Sheet 3 of 4)

Parameter/Icon Description
Chemical Formula • For a peptide, select Peptide, enter the peptide sequence using the one-letter
...continued symbols for the amino acid residues (see One- and Three-Letter Abbreviations for
Amino Acid Residues), select the ionization species, and type or select the charge of
the adduct ion of interest.
Example: For Hexarelin (a hexapeptide), use the one-letter code—HWAWFK.

When you click Apply, the Trace Type changes to Mass Range, and the mass-to-charge
ratio of the specified ion appears in the Ranges column.
Mass Tolerance Entering a chemical formula in the Chemical Formula column or selecting Mass Range
in the Trace Type column makes this table cell available.

Specify the mass tolerance for the mass-to-charge ratio of the specified ion.

Range: 0.00 to 10.00; default: 0.5 amu

Delay Time (hidden by Specify a delay time (in minutes) to shift trace 1 (Trace Type column) so that it aligns
default) with trace 2 (Trace Type2 column). A positive delay time shifts trace 1 to higher
retention times by this amount.

Range: –5.00 to 5.00; default: 0.00

Reference Select this check box to make the chromatogram trace a reference trace. A reference
trace is always displayed in the Chromatogram view so that you can compare other
traces to it.
Plot Operator Select a plot operator, either + (add) or – (subtract).
• When you select +, the Chromatogram view displays trace 1 plus trace 2.
• When you select –, the Chromatogram view displays trace 1 minus trace 2.
For example, from a chromatogram you can use the subtract (–) plot operator to
subtract contributions from a solvent or from other noise.
For MS detectors, these are the trace 1 and trace 2 combinations:
• mass range ± mass range
• mass range ± base peak
• base peak ± mass range
• base peak ± base peak
• TIC – mass range
• TIC – base peak

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Table 19. Chromatogram Ranges view parameters and icons (Sheet 4 of 4)

Parameter/Icon Description
Plot Operator ...continued For PDA detectors, these are the trace 1 and trace 2 combinations:
• wavelength range ± wavelength range
• wavelength range ± spectrum maximum
• spectrum maximum ± wavelength range
• spectrum maximum ± spectrum maximum
• total scan – wavelength range
• total scan – spectrum maximum
Trace Type2 (hidden by Select the type of trace to add to or subtract from trace 1.
default)
Range2 Specify the wavelength or mass range for trace 2.
Comment Specify a comment for the row.
Icon
Fill Down Opens the Fill Down dialog box for populating multiple rows with duplicate parameter
settings.

For details, see Using the Fill Down Feature.

Field Chooser Displays the Field Chooser dialog box for selecting which columns appear in the
Chromatogram Ranges view.

For details, see Selecting the Columns to Display in a View or Dialog Box with Tabular
Data.
Check boxes in the Display Select the check box to add the defined chromatogram to the Chromatogram view.
column Selecting the check box in the last row populates the row with the settings from the
previous row.

You can edit the File Name parameter to change the new entry to that raw data file, or
click at the end of the file name to browse to and select a new raw data file.

Using the Fill Down Feature

To specify several similar chromatogram traces, use the Fill Down feature in the
Chromatogram Ranges view. With this feature, you can copy the selected parameter values
from the currently selected row to other specified rows.

Y To fill down data

1. In the Chromatogram Ranges view, select the trace row that you want to copy data from.
2. Click the Fill Down icon, .
The Fill Down dialog box opens (Figure 57).
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Figure 57. Fill Down dialog box

3. Do one of the following:

• Select the check box for each parameter value that you want to fill down.
• Click All to select all the check boxes.
–or–
• Click Clear to clear all the check boxes.
4. In the Fill Rows boxes, type the starting and ending row numbers, and then click OK.

The application copies the selected parameter values from the currently selected row and
pastes them into the specified starting and ending rows. If a value is invalid, it appears in red.

Moving a Row
You can move a row in the Chromatogram Ranges view by dragging it to the new location.

Y To move a row

1. Place the pointer in an area somewhere in the row that you want to move, but not inside
an editable field and not in the blank area to the left of the row.
For example, place the pointer in the area next to the Display check box (Figure 58).

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Figure 58. Example pointer location to move a row

Place the pointer in this area.

2. Drag the row up or down to move it to the new location.

As you drag the row above or below another row, that other row temporarily turns red.
After you drop the row, it appears above or below the red row.

Deleting Rows
You can delete rows in the Chromatogram Ranges view. For each row that you delete, the
application automatically deletes the corresponding trace in the Chromatogram view.

Note When the view has only one row, you cannot delete the row.

Y To delete rows in the Chromatogram Ranges view

1. Select the rows that you want to delete as follows:

• To select a single row, click the arrow icon, , that appears when you point to the
left of the Display column.
• To select adjacent rows, drag the pointer across the group of rows (in the column to
the left of Display column) or use the SHIFT key.
• To select nonadjacent rows, press the CTRL key and click to the left of the Display
column for each row.
Selected rows are highlighted in blue.
2. Press the DELETE key and click Yes in the confirmation dialog box to remove the
selected rows.

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Peaks List View

The Peaks List view lists the chromatogram peaks that the peak detection algorithm
automatically finds and also lists the peaks that you manually add.

Y To display the Peaks List view

• Apply the ICIS, Genesis, Avalon, or PPD peak detection algorithm (see
Automatically Detecting and Integrating Chromatographic Peaks).

IMPORTANT Applying the peak detection algorithm the first time automatically
opens the Peaks List view. However, if you then close this view and apply the peak
detection algorithm again, the Peaks List view does not automatically reopen. To
manually open this view, click the Workspace Processing tab to display the
Workspace Processing toolbar, and then click Peak List in the Workspace
Processing toolbar.

• Manually add a peak (see Manually Adding, Undoing, and Deleting

Chromatographic Peaks).
–or–
a. Click the Workspace Processing tab to display the Workspace Processing toolbar.
b. Click Peaks List.

Figure 59 and Figure 60 show examples of the Peaks List view. In Figure 60, the Name
column is populated with the compound name specified in the instrument method for the
selected scan filter. The table values are read-only.
Figure 59. Peaks List view
Detected peaks

Peaks List view

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Figure 60. Peak List view with a populated Name column

Compound name Compound name

specified in the in the Peaks List
Instrument Method for
a specific scan filter

Table 20 describes the parameters and icons for the Peaks List view.
Table 20. Peaks List view parameters and icons (Sheet 1 of 2)
Parameter Description
Reset Restores the data display in the Chromatogram view to the full range of the x axis and y axis
after you zoom in by clicking a row in the peaks list.

Field Chooser Displays the Field Chooser dialog box, where you select which fields appear in the peaks list
(see Selecting the Columns to Display in a View or Dialog Box with Tabular Data).

Elemental Select to run the elemental composition algorithm. This algorithm requires full-scan data.
Composition
Field
Index Unique identification number for each chromatogram peak. The index increments from lowest
to highest retention time in a trace and from the top trace to the bottom trace.

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Table 20. Peaks List view parameters and icons (Sheet 2 of 2)

Parameter Description
Name Compound name from the instrument method (Figure 60).
Note For some Thermo Scientific mass spectrometers, for example, the triple quadrupole
product line, the instrument method that you use for data acquisition includes a table where
you can associate a compound name with each scan filter.
When you view the scan filters on the Scan Filters page, the associated compound names
appear next to the scan filter. You cannot edit the names in the FreeStyle application.
RT (Min) Retention time (in minutes) corresponding to the apex of the peak.
RT (Sec) Retention time (in seconds) corresponding to the apex of the peak.
Start RT Retention time corresponding to the start of the chromatographic peak, where the detection
signal increases beyond the threshold criteria.
End RT Retention time corresponding to the end of the chromatographic peak, where the detection
signal decreases below the threshold criteria.
Base Peak Mass-to-charge ratio of the most abundant ion at the apex of the peak. The Peak List view
displays 0.00 if no mass spectral data is present.
Peak Area Area of the peak in units of counts × seconds. Displayed in either general or exponential format
(see Labeling Chromatogram Peaks or Local Maxima).
Peak Height Number of counts at the peak apex. Displayed in either general or exponential format (see
Labeling Chromatogram Peaks or Local Maxima).
Baseline Width Difference (in minutes) between the start and end retention times.
Signal To Noise Signal-to-noise ratio of the integrated peak.
Empirical Formula Chemical formula determined by the elemental composition algorithm.

By default, this column is hidden. Selecting the Empirical Formula check box adds the column
to the table and runs the elemental composition algorithm.
File Name Displays the path and name of the raw data file.
To display the Open Raw File dialog box, click the browse icon, , at the end of the file name.
Scan Filter Select a filter to apply to the raw data file. The application autosenses the metafilters (including
SRM or compound filters) in the raw data file in this order: MS, MS2, ETD, HCD, MSN,
and then the individual scan filter list.
For grouped filters, the Filter list displays only the first filter in the group. The Spectrum view
displays the actual filter for the scan. For more information, see Scan Filter Parameters.
Trace ID Number that identifies the chromatogram trace. The Trace ID increments from the top trace
to the bottom trace.
% Area Peak area as a percentage of the total peak area.
% Height Peak height as a percentage of the total peak height.
Integration Method Displays the integration method: ICIS, Genesis, Avalon, PPD, or manual.

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Chromatogram-specific Pages in the Info Bar

Before you open a workspace in the FreeStyle window, the Info Bar contains only the Isotope
Simulation page (see Isotope Simulation Page). Opening a raw data file adds the Peak
Detection, MSn Browser, and Detector Type pages to the Info Bar; whereas, opening a
sequence file opens these pages and the Sequence Files page. The Detector Type page lists the
detectors used to acquire data for the active raw data file and the MS Trending selection if the
raw data file contains mass spectrometry data.

Note When you click a row in the Filter or Trace Type columns in the Chromatogram
view, the application replaces the Detector Type page with the Scan Filters page or the
Trace Type page, respectively.

These are the pages in the Info Bar:

• Detector Type Page
• Trace Type Page
• Scan Filters Page
• Peak Detection Page
• Genesis Peak Detection Page
• ICIS Peak Detection Page
• Avalon Peak Detection Page
• PPD Peak Detection Page
• Sequence File Page

Detector Type Page

The detector type determines what types of traces the Chromatogram view displays. Use the
Detector Type page of the Info Bar to select the detector type. The detector types shown on
this page are the same as those in the Detector Type list in the Chromatogram Ranges view.

These are the possible detector types:

• MS—Mass spectrometry data
• A/D Card—Data from an analog-to-digital converter
• MS Trending—Instrument status readings for the mass spectrometer
• UV—Data from an analog detector
• PDA—Wavelength scan data from a photodiode array detector

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Y To display the Detector Type page

Click the Detector Type tab in the Info Bar.

Figure 61 shows a typical Detector Type page.
Figure 61. Detector Type page

Y To display a plot in the Chromatogram view

On the Detector Type page, do one of the following:

• To display an unfiltered plot of the MS data, click MS.
• To display a total scan plot for the PDA data, click PDA.
• To display a plot for a UV channel, click UV.
• To display a plot for the data acquired with an analog-to-digital converter, click A/D
Card.
• To display a plot for one of the MS detector’s status readbacks, click MS Trending,
click a row in the Trace Type column in the Chromatogram Ranges view, and then,
on the Trace Type page, click a trace type (instrument readback).

Trace Type Page

Use the Trace Type page to select the type of trace that the Chromatogram view displays. The
available trace types depend on which detector type you select on the Detector Type page:
• For MS detectors, choose the TIC, mass range, base peak, neutral fragment, or mass
defect filter trace type.
• For PDA detectors, choose the wavelength range, total scan, or spectrum maximum trace
type.
• For analog detectors, choose the analog 1, 2, 3, or 4 trace type.
• For MS Trending, choose any of the instrument status selections.
The trace types that the Trace Types page lists are the same as in the Trace Type list in the
Chromatogram Ranges view.

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Y To display the Trace Type page

1. Open the Chromatogram Ranges view.

2. Click the dropdown arrow in the Trace Type column.

Figure 62 shows trace types for an MS detector type.

Figure 62. Trace Type page

Scan Filters Page

Use the Scan Filters page to select the type of scan filter, if any, that the application applies to
the raw data file. The filter types on the Scan Filters page are the same as in the Filter box
dropdown list in the Chromatogram Ranges view.

For instructions on using the Scan Filters page, see Using the Scan Filters Page to Display a
Filtered Chromatogram.

Note For grouped filters (MS, MS2, HCD, and so on), the Scan Filters page displays only
the first filter in the group. The scan header in the Spectrum view displays the actual filter
for the scan.

Y To display the Scan Filters page in the Info Bar

In the Workspace Options toolbar, in the Filter area, click Scan Filters.

Figure 63 shows a Scan Filters page.

Figure 63. Scan Filters page

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Table 21 describes the parameters on the Scan Filters page.

Table 21. Scan Filters page parameters
Parameter Description
Filter Parameters
Time Range (min) Specifies the time range to display in the filter list. Filters outside
the specified time range do not appear in the filter list. The
specified time range must be within the retention time range for
the data file.

Use an asterisk to specify the full retention time range.

Track Specifies the time range by dragging the pointer across the Time
axis in the Chromatogram view.
Filter list
<No Filter> Displays an unfiltered chromatogram.
MS Displays only full-scan MS data.
MSn (where n = 2 or Displays MS/MS data of the selected MS/MS order only.
higher)
Scan filters Displays a chromatogram for a specific scan filter.
Buttons
Export Copies the file name of the raw data file and the contents of the
Scan Filters page to the Clipboard.
Print Opens the Print dialog log box for selecting the printer and print
preferences for printing the contents of the Scan Filters page.
Help Opens the Help to the Scan Filters page topic.

Peak Detection Page

Use the Peak Detection Page to identify and integrate the chromatogram peaks in an active
chromatogram plot. Specify a peak detection algorithm that the application applies to detect
the chromatogram peaks in the active chromatogram plot. For instructions on using the Peak
Detection Page, see Automatically Detecting and Integrating Chromatographic Peaks.
Figure 64 shows the parameters for the peak identification and detection algorithms.

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Figure 64. Peak Detection Info Page

Table 23 describes the parameters for the peak identification and detection algorithms.
Table 22. Peak Identification and Detection algorithm parameters
Parameter Description
Identification
Returned Peaks Type of peak detection results to be returned:
• All Peaks: Displays all the peaks based on the selected
algorithm for component identification.
• Nearest Peak: Displays the peak with the nearest
retention in the chromatogram within the specified
retention time window for component identification.
• Highest Peak: Displays the peak with the highest
retention in the chromatogram within the specified
retention time window for component identification.
Expected Time (min) Expected retention time for the component. Available only
when you select the Nearest or Highest peak.
Window (sec) Retention time window for the component. Available only
when you select the Nearest or Highest peak.
Component Name Specifies the component name used for processing.
Automatically imports the component name when a
processing method is imported to the trace.

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Table 22. Peak Identification and Detection algorithm parameters

Parameter Description
Peak Detection Algorithm
Selected Algorithm Specifies the peak detection algorithm to be used for
processing the chromatographic peaks:
• Genesis—Use for mass spectrometry data.
• ICIS—Use for mass spectrometry data.
• Avalon—Use for UV or PDA data. This algorithm
detects negative chromatographic peaks more
accurately than the ICIS or Genesis algorithms.
• PPD—Use to minimize the number of optimization
parameters.

Genesis Peak Detection Page

The Genesis peak detection algorithm is the original Thermo Xcalibur peak detection
algorithm and has been provided for backward compatibility with Xcalibur 1.0 data system
studies. Use the Genesis Peak Detection page of the Info Bar to specify peak identification and
peak integration criteria that the Genesis algorithm applies toward chromatograms in the
Chromatogram view.

For information about using the Genesis peak detection algorithm, see Automatically
Detecting and Integrating Chromatographic Peaks.

Y To display the Genesis Peak Detection page

1. Click the Peak Detection tab in the Info Bar.

2. In the list, select Genesis.

Figure 65 shows the parameters for the Genesis peak detection algorithm.

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Figure 65. Genesis Peak Detection page

Table 23 describes the parameters for the Genesis Peak Detection page.
Table 23. Genesis peak detection parameters (Sheet 1 of 4)
Parameter Description
Application of Settings
Apply to All Plots Apply the peak identification and integration settings to all
displayed chromatograms.

To apply the criteria to only the active chromatogram, clear this

option.

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Table 23. Genesis peak detection parameters (Sheet 2 of 4)

Parameter Description
Peak Parameters
Percent of Highest Specify a percentage threshold to limit the number of peaks to be
Peak submitted for further processing. The algorithm discards any
detected peak that has an intensity less than the threshold
percentage of the most intense peak.

Range: 0.0–100.0; default: 10.0

Minimum Peak Ht Specify the peak signal-to-noise (S/N) value to equal or exceed as a
(S/N) criterion for peak detection. The algorithm ignores all
chromatogram peaks that have S/N values less than the minimum
peak height S/N value.

Range: 1.0 (all peaks) to 999.0; default: 2.0

S/N Threshold Specify the threshold for detecting peak edges. The algorithm
calculates the S/N ratio by using only the baseline signal. It
excludes any extraneous or minor detected peaks from the
calculation.

Range: 0.0–999.0; default: 2.0

Valley Detection Select the valley detection approximation method to detect
Enabled unresolved peaks. This method drops a vertical line from the apex
of the valley between unresolved peaks to the baseline. The
intersection of the vertical line and the baseline defines the end of
the first peak and the beginning of the second peak.
Expected Width (sec) Specify the expected peak width (in seconds). This value controls
the minimum width that a peak must have if you selected the
Valley Detection Enabled option.

With valley detection selected, the algorithm ignores any valley

points nearer than the expected width/2 to the top of the peak.
When it finds a valley point outside the expected peak width, the
algorithm terminates the peak at that point. The algorithm always
terminates a peak when the signal reaches the baseline,
independent of the value set for the expected peak width.

Range: 0.0–999.0; default: 0.0

Constrain Peak Width Limits the peak width of a component during peak integration. To
control when peak integration is turned on and off, specify a peak
height threshold and a tailing factor.

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Table 23. Genesis peak detection parameters (Sheet 3 of 4)

Parameter Description
Peak Ht (%) Specify the peak height (as a percentage) where the algorithm tests
the width of target peaks.

Range: 0.0–100.0; default: 5.0

Tailing Factor Specify a factor that controls how the algorithm integrates the tail
of a peak. This factor is the maximum ratio of the trailing edge to
the leading edge of a constrained peak.

Range: 0.5–9.0; default: 1.0

Advanced
RMS Calculate noise as a root-mean-square (RMS) value.
Peak to Peak Calculate noise as a peak-to-peak value.
Manual Noise Region Specify a region of the chromatogram that the algorithm uses to
determine noise. See Selecting the Manual Noise Region for the
Genesis and ICIS Algorithms.
RT Range Specify the RT range that the algorithm uses to determine noise.
The RT range must be within the chromatogram range.
Baseline Noise Specify a percentage value that controls how the algorithm draws
Tolerance (%) the baseline in the noise data. The higher the baseline noise
tolerance value, the higher it draws the baseline through the noise
data.

Range: 0.0–100.0; default: 10.0

Min Number of Scans Specify the minimum number of scans that the algorithm uses to
in Baseline calculate a baseline. A larger number includes more data in
determining an averaged baseline.

Range: 2–100; default: 16

Baseline Noise Specify the baseline noise rejection factor. This factor controls the
Rejection Factor width of the RMS noise band above and below the peak detection
baseline. The algorithm applies this factor to the raw RMS noise
values to raise the effective RMS noise that the algorithm uses
during peak detection. It responds by assigning the left and right
peak boundaries above the noise and, therefore, closer to the peak
apex value. This action effectively raises the peak integration
baseline above the RMS noise level.

Range: 0.1–10.0; default: 2.0

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Table 23. Genesis peak detection parameters (Sheet 4 of 4)

Parameter Description
Peak S/N Cutoff Specify the S/N level that the algorithm defines as the top of the
peak edge. For example, if the S/N level at the apex is 500 and the
Peak S/N Cutoff value is 200, the algorithm defines the right and
left edges of the peak when the S/N reaches a value less than 200.

Range: 50.0–10 000.0; default: 200.0

Rise Percentage Specify how much above the baseline the trace can rise (as a
percentage) after passing through a minimum inflection point in
the trace (before or after the peak).

If the trace exceeds this value, the algorithm applies valley

detection peak integration criteria. It applies this test to both the
left and right edges of the peak. The rise percentage criterion is
useful for integrating peaks with long tails.

Range: 0.1–500.0; default: 10.0

Valley S/N Specify the S/N criterion that the algorithm uses for valley
detection.

Range: 1.0–100.0; default: 1.0

Background Specify the background recomputation interval (in minutes). The
Recomputation (min) algorithm periodically recalculates the representative background
scan it uses for background subtraction to compensate for the
possibility that the composition of the background might change
over the course of a run. The background recomputation interval is
the time interval between these recalculations.

Range: 0.5–10.0; default: 5.0

Number of Scans in Specify the number of background scans the algorithm uses to
Background determine the background.

Range: 2–100; default: 5

Button
Apply Starts peak detection by using the Genesis peak detection
algorithm.
Save As Defaults Saves the current settings as default settings. After you save the
settings as defaults, you can restore these values at any time by
clicking Load Default.
Load Defaults Restores the current default settings.
Help Opens the FreeStyle Help to the Genesis peak detection topic.

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ICIS Peak Detection Page

The ICIS peak detection algorithm is the default algorithm that was designed for MS data. It
is known for peak detection efficiency at low MS signal levels.

Use the ICIS Peak Detection page of the Info Bar to specify peak detection and integration
criteria. The application applies the peak detection algorithm to the active plot or all the plots
in the Chromatogram view.

For more information, see Automatically Detecting and Integrating Chromatographic Peaks.

Y To display the ICIS peak detection parameters

1. Click the Peak Detection tab in the Info Bar.

2. In the list, select ICIS.

Figure 66 shows the parameters for the ICIS peak detection algorithm.
Figure 66. ICIS Peak Detection page

Table 24 describes the parameters for the ICIS peak detection algorithm.

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Table 24. ICIS Peak Detection page parameters (Sheet 1 of 2)

Parameter Description
Application of Settings
Apply to All Plots Apply the current chromatogram peak identification and integration
settings to all displayed plots.

To apply the criteria to only the active plot, clear this option.
Peak Parameters
Baseline Window Specify the number of scans, from the apex down each side of the
peak, that the peak detection algorithm uses to determine the
minimum baseline for the peak. A higher number of scans means a
wider scan range, resulting in a lower baseline.

Range: 1–500; default: 40

Area Noise Factor Specify the noise level multiplier that the peak detection algorithm
uses to determine the peak edge after the location of the possible
peak.

Range: 1–500; default: 500

Peak Noise Factor Specify the noise level multiplier that the peak detection algorithm
uses to determine the potential peak signal threshold.

Range: 1–1000; default: 10

Constrain Peak Limits the peak width of a component during the peak integration
Width of a chromatogram. You can then set values that control when peak
integration turns on and off by specifying a peak height threshold
and a tailing factor.
Peak Ht (%) Specify how much above the baseline (as a percentage) a signal must
be of the total peak height before integration turns on or off.

Range: 0.0–100.0; default: 5.0

Tailing Factor Specify a factor that controls how the algorithm integrates the tail of
a peak. This factor is the maximum ratio of the trailing edge to the
leading edge of a constrained peak.

Range: 0.5–9.0; default: 1.0

Advanced
Manual Noise Region Specify a region of the chromatogram that the peak detection
algorithm uses to determine noise. You can enter the retention time
(RT) in the RT Range box. See Selecting the Manual Noise Region
for the Genesis and ICIS Algorithms.

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Table 24. ICIS Peak Detection page parameters (Sheet 2 of 2)

Parameter Description
RT Range Specify the RT range that the algorithm uses to determine noise.
The RT range must be within the chromatogram range.
INCOS Noise Use a single pass algorithm to determine the noise level. For more
information, see ICIS Signal-to-noise Estimation.
Repetitive Noise Use a multiple pass algorithm to determine the noise level. In
general, this algorithm is more accurate in analyzing the noise than
the INCOS Noise algorithm; however, it might take longer
depending on the data. For more information, see ICIS
Signal-to-noise Estimation.
RMS Calculate noise as root mean square (RMS). By default, the
algorithm uses peak-to-peak for the noise calculation. When you
determine the noise region manually, the algorithm automatically
selects RMS.
Min Peak Width Enter the minimum number of scans required in a peak.

Range: 0–100; default: 3

Multiplet Resolution Specify the minimum separation in scans between the apexes of two
potential peaks. This criterion determines if two peaks are resolved.

Range: 1–500; default: 10

Area Tail Extension Specify the number of scans past the peak endpoint to use in
averaging the intensity.

Range: 0–100; default: 5

Area Scan Window Specify the number of scans on each side of the peak apex to include
in the area integration.

Range: 0–100; default: 0

Note Specifying zero scans means that the algorithm includes all
scans in the area integration, from the peak start to the peak end.
Button
Apply Starts the ICIS peak detection algorithm.
Save As Defaults Saves the current settings as the default settings.
Load Defaults Restores the current default settings.
Help Opens the FreeStyle Help to the ICIS peak detection topic.

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ICIS Signal-to-noise Estimation

ICIS peak detection uses one of two algorithms for signal-to-noise ratio (SNR) estimation:
• INCOS Algorithm (single pass algorithm)
• Repetitive Algorithm (multiple pass algorithm)

See also, Baseline in ICIS.

INCOS Algorithm
For a chromatogram of intensities—I(n)—(where n is a retention time position from 1 to N,
which does not take into account time spacing), the application calculates a noise value for
each point (n=2 to N–1) as follows:

noise n abs I n – 1 - 2*I n In 1

This is a triangular method in which the noise for every point is estimated by using only its
direct neighbors. It assumes that direct neighbors should have a very similar intensity. If the
three points have equal intensity, the estimated noise will be zero. If two of the points have
equal or very similar intensity and the other point has an intensity difference of ΔI, the
estimated noise will be approximately ΔI.

The noise for the chromatogram is then the average of all noise(n), such that:

Repetitive Algorithm
The repetitive algorithm is similar to the INCOS algorithm; the application calculates a local
noise value for each data point using the same method used for the INCOS algorithm.

The application saves all local noise/intensity value pairs in an array. The decision whether a
local noise event is accepted or not is postponed until all noise events are recorded (unlike
INCOS where only events smaller than 8 times the square root of the minimum of the
current three scans are accepted as noise).

The starting value for the noise is the average of all local noise events.

In each iteration (maximal 10), the local noise values of the intensities that are bigger than the
average intensity of all remaining noise events are discarded and a new average noise is
calculated. If the new average noise is smaller than old_noise / 1.1, the application performs
another iteration; if not (or if there were already 10 iterations), the process is finished.

In a final step, the application discards the most intense third and the least intense third of the
remaining values and uses the remaining values to calculate the noise.

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Baseline in ICIS
The application calculates noise in ICIS II similar to the INCOS algorithm. The baseline
estimation is trivial: a sum of intensities of all scans, which are counted for INCOS noise,
divided by the number of those scans.

The baseline correction works as follows:

1. For each scan x in the chromatogram, the application determines whether the scan in
question is a baseline scan or not (the first and last scan in the display area are always
baseline scans).
2. For the remaining x scans, a baseline scan is available if a line can be placed in the
chromatogram using the point (Scan x / Intensity of Scan x), so that the intensities of all
neighboring right and left scans come to lie above the straight line.
3. After all baseline scans have been determined, the intensities between the baseline scans
are corrected (an amount is deducted from the intensity values between the baseline scans,
which results from the Y value of a straight line through the neighboring baseline scans at
the scan (x) point.
4. The intensities of all baseline scans are set to zero.
5. After the (optional) smoothing and the baseline correction (optional in CHRO), the
actual peak detection takes place:
The criterion for peak detection is an intensity threshold ITR, which is determined as
follows:
• For baseline corrected chromatograms: ITR = nm * Noise (nm = Noisemultiplier)
• For uncorrected chromatograms: ITR = baseline + nm * noise

Avalon Peak Detection Page

The Avalon peak detection algorithm supports detectors other than mass spectrometers and
detects negative chromatographic peaks more accurately than the Genesis or ICIS peak
detection algorithms. Use the Avalon Peak Detection page in the Info Bar to specify peak
identification and integration criteria. You can apply this algorithm to the active plot or all the
plots in the Chromatogram view.

Y To display the Avalon Peak Detection page

1. Click the Peak Detection tab in the Info Bar.

2. Select the Avalon search algorithm.

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Figure 67 shows the parameters for the Avalon peak detection algorithm.
Figure 67. Avalon Peak Detection Settings page

Table 25 describes the parameters for the Avalon peak detection algorithm.
Table 25. Avalon Peak Detection page parameters (Sheet 1 of 4)
Parameter Description
Application of Settings
Apply to All Plots Apply the current chromatogram peak identification and integration
settings to all displayed chromatogram plots.

To apply the criteria to only the active plot, clear this check box.

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Table 25. Avalon Peak Detection page parameters (Sheet 2 of 4)

Parameter Description
Event List
Event List Specify the settings for initial events and user-defined timed events
in the event list.

To calculate values for initial events, click Auto Calculate Initial

Events.

To change the settings in the event list, highlight the row and then
enter the revised settings in the boxes below the list. Click Add to
add a new row of entered values to the event list. Click Change to
update automatically both the event list and the chromatogram
display.

There are seven permanent integration events, identified by the

Initial Value setting in the Time column. These are the default
integration events that the Avalon integration algorithm requires.
You can change the value of an initial entry integration event, but
you cannot delete it or change its time value.
Event List Entry
Time Specify the value for the Time column for the highlighted entry in
the event list.

You cannot change the time entry for initial value events.

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Table 25. Avalon Peak Detection page parameters (Sheet 3 of 4)

Parameter Description
Event Select the event type from these options:
• Start/End Threshold: Directly related to the RMS noise in the
chromatogram, this value is the start or end threshold, the
fundamental control used for peak detection.
• Area Threshold: Controls the area cutoff. The algorithm does
not detect any peaks with a final area less than the area
threshold. This control is in units of area for the data.
• P-P Threshold: The peak-to-peak resolution threshold controls
how much peak overlap must be present before two or more
adjacent peaks create a peak cluster. Peak clusters have a baseline
drop instead of valley-to-valley baselines. This option is specified
as a percentage of the peak height overlap.
• Negative Peaks: Allows or denies negative peaks.
• Bunch Factor: The number of points that are grouped together
during peak detection. It controls the bunching of
chromatographic points during integration and does not affect
the final area calculation of the peak. The Bunch Factor must be
an integer between 1 and 6; a high bunch factor groups peaks
into clusters.
• Tension: Controls how closely the baseline must follow the
overall shape of the chromatogram. A lower baseline tension
traces the baseline to follow changes in the chromatogram more
closely. A higher baseline tension follows the baseline less closely
over longer time intervals. This option is specified in minutes.
• Tangent Skim: For fused peaks that are significantly different in
size, the tangent skim method allocates area to the various peaks.
By default, the algorithm chooses the tallest peak in a cluster as
the parent. You can also identify which peak in the cluster is the
parent. The algorithm detects tangent skim peaks on one or
both sides of the parent peak. Tangent skim automatically resets
at the end of the peak cluster.

The threshold and bunch factor parameters are the most important
ones in controlling peak detection.
Value Specify the value for Value column for the currently highlighted
entry in the event list. The range of factors allowed for each value is
specific to each event.

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Table 25. Avalon Peak Detection page parameters (Sheet 4 of 4)

Parameter Description
Button
Auto Calc Initial Searches for the best values of initial events that detect peaks in the
Events data. This button is active if you have a raw data file open. The
Avalon algorithm automatically estimates the initial values for the
detection of peaks based on the data in the current raw data file, and
then displays those initial values in the event list. Any timed event in
the event list is unchanged.

Automatic calculation of initial events determines initial values for

the following events only: start threshold, end threshold, area
threshold, P-P [resolution] threshold, bunch factor, negative peaks,
and tension. Additionally, you can specify timed events for these
events in the same event list.
Add Add a row to the event list. When you click Add, both the event list
and the chromatogram update automatically with the added
specification in the currently selected chromatogram.
Delete Removes a highlighted event from the event list. You cannot delete
initial values.
Change Updates a highlighted entry in the event list. When you click
Change, peak detection with the updated specification occurs
automatically for the currently selected chromatogram. For initial
events, the algorithm changes only the values, and not the events.
Help Opens the FreeStyle Help to the Avalon peak detection topic.

PPD Peak Detection Page

The PPD peak detection algorithm fits the data to model peak functions.

Use the PPD Peak Detection page of the Info Bar to specify peak detection criteria. The
application applies the peak detection algorithm to the active raw data file displayed in the
FreeStyle window.

For information about using the PPD peak detection algorithm, see Automatically Detecting
and Integrating Chromatographic Peaks.

Y To display the PPD Peak Detection page

1. Click the Peak Detection tab in the Info Bar.

2. Select the PPD algorithm.

Figure 68 shows the settings for the PPD peak detection algorithm.

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Figure 68. PPD Peak Detection page

For information about adding peak labels, see Labeling Chromatogram Peaks or Local
Maxima.

Table 26 describes the parameters for the PPD peak detection algorithm.
Table 26. PPD Peak Detection page parameters (Sheet 1 of 2)
Parameter Description
Application of Settings
Apply to All Plots Applies the current chromatogram peak identification and
integration settings to all displayed plots.

To apply the criteria to only the active plot, clear this option.
Peak Parameters
Signal To Noise Specifies the absolute signal-to-noise threshold. A value of 0 applies
no filtering.

Range: 0.00–100.00; default: 1.0

Merge Overlapping (Default) Merges overlapping peaks into one peak. Use this option
for LC/MS data to avoid reporting small shoulders as separate peaks.
Resolve Resolves overlapping or touching peaks into two chromatographic
peaks. Use this option for GC/MS data.
Merge Touching Merges two peaks whose edges barely overlap at the 1% intensity
level.
Button
Apply Starts the PPD peak detection algorithm.
Save As Defaults Saves the current settings as the default settings.

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Table 26. PPD Peak Detection page parameters (Sheet 2 of 2)

Parameter Description
Load Defaults Restores the current default settings.
Help Opens the FreeStyle Help to the PPD peak detection topic.

The PPD peak detection algorithm automatically resolves all the chromatographic peaks, and
selecting the Resolve option displays all the detected peaks (see Figure 69).
Figure 69. PPD peak detection with the Resolve option

By default, the Merge Overlapping option is selected. For LC/MS data, use Merge
Overlapping to avoid reporting small shoulders as separate peaks. Compare Figure 70 where
the overlapping peaks are merged and reported as two peaks to Figure 69 where all the peaks
are resolved and reported as seven separate peaks.

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Figure 70. PPD peak detection with the Merge Overlapping option

Sequence File Page

The Sequence File page of the Info Bar displays a sequence of raw data files that you created or
opened. Click a raw data file in the list to display its trace in the Chromatogram view.

Y To display the Sequence File page

Create or open a sequence of raw data files.

For more information, see Using Workspace Pages or Working with Sequences.

Figure 71 shows a typical Sequence File page.

Figure 71. Sequence File page

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 4

Reviewing Spectral Data

Use the spectra-specific toolbars, views, and Info Bar pages of the FreeStyle application to
review spectral data from the mass spectrometer or PDA detector.

To display spectra of interest, follow the procedures in these topics.

Contents
• Displaying the Scan for a Time Point in a Chromatogram
• Adding Spectrum Views to the Workspace
• Selecting Spectra from the Spectrum Ranges Dialog Box
• Creating a MultiSpectrum View and Changing Its Spectrum Plots
• Formatting a MultiSpectrum View
• Linking and Releasing Spectra to and from a Chromatogram
• Averaging Spectra
• Subtracting Background Spectra
• Exporting a Selected Spectrum to a New Raw Data File
• Using Precursor Markers to Open Data-Dependent Scans
• Selecting Spectra from an MSn Tree
• Specifying the Display Options for a Spectrum View
• Changing the Zoom Level in a Spectrum View or MultiSpectrum View
• Spectra-specific Toolbars
• Spectra-specific Views
• Spectrum Ranges Dialog Box
• MSn Browser Page

Note For information about scan headers and custom annotations, see Scan Headers and
Scan Header Abbreviations and Adding Text, Graphic, and Structure Annotations to a
Graphical View, respectively.

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Displaying the Scan for a Time Point in a Chromatogram

Displaying the Scan for a Time Point in a Chromatogram

Follow this procedure to display the scan for a particular time point of a chromatogram.

Y To display the scan corresponding to a particular time point in a chromatogram

1. Display the chromatogram plots of interest as described in Defining a Chromatogram

Trace from the Chromatogram Ranges View.
2. Make sure that the Spectrum view or MultiSpectrum view links to the chromatogram.

Note The text appended to a Spectrum view indicates its linkage status. For example,
the text C1T1 at the end of a Spectrum view’s name indicates that actions performed
on trace 1 in Chromatogram view 1 affect the spectrum displayed.

3. Click the chromatogram plot at the retention time or scan number that you want to view.
In the Chromatogram view, a red vertical marker, , indicates the selected data point.
The Spectrum view displays the spectrum for that retention time or scan number. On the
computer keyboard, use the right or left arrow keys to increment or decrement the scan
number.

Note When you do not apply a scan filter, the scan number increments and
decrements by one. When you do apply a scan filter, the scan number increments and
decrements to the next scan number that meets the filter criteria.

Adding Spectrum Views to the Workspace

You can add multiple Spectrum views and MultiSpectrum views to the workspace.

Y To add a Spectrum view

1. Open a raw data file (see Opening Raw Data Files or Sequence Files).
With the factory default layout, the Chromatogram view appears at the top and the
Spectrum view appears at the bottom and displays scan #1.
2. To add another Spectrum view to the workspace, do one of the following:
• In the Workspace Options toolbar, click Spectrum.
A copy of the active spectrum appears in the new Spectrum view.
• Open the MSn Browser page and select the fragmentation spectra of interest (see
Selecting Spectra from an MSn Tree).
A new Spectrum view opens for each selected spectrum.

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Selecting Spectra from the Spectrum Ranges Dialog Box

For information about displaying a different spectrum in the Spectrum view, see these topics:
• Displaying the Scan for a Time Point in a Chromatogram
• Selecting Spectra from the Spectrum Ranges Dialog Box

Selecting Spectra from the Spectrum Ranges Dialog Box

You can use the Spectrum Ranges dialog box to select the spectrum to be displayed in a
Spectrum view or the spectra to be displayed in a MultiSpectrum view. For information about
creating an average spectrum or performing background subtraction, see these topics:
Creating an Average Spectrum from the Spectrum Ranges Dialog Box or Using the Spectrum
Ranges Dialog Box to Subtract Background Spectra.

Note When a spectrum is linked to a chromatogram, you cannot select a different raw
data file, detector, or scan filter from the Spectrum Ranges dialog box.

Y To select spectra with the Spectrum Ranges dialog box

1. Do one of the following:

• Click either a Spectrum view or a MultiSpectrum view to select it. Then, in the
Workspace Options toolbar, click Spectrum and choose Ranges from the dropdown
menu.
–or–
• Right-click a Spectrum view or a MultiSpectrum view and choose Ranges.
The Spectrum Ranges dialog box opens with a list of the scans currently displayed in the
selected view (Figure 72).
Figure 72. Spectrum Ranges dialog box

2. Do any of the following:

• To add another spectrum plot, select the last check box in the Display column.
• For a MultiSpectrum view, to remove a spectrum plot, clear its associated check box.

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Creating a MultiSpectrum View and Changing Its Spectrum Plots

• (Released spectrum only) From the File Name list, browse to and select a different
raw data file.
• (Released spectrum only) From the Detector Type list, select a different detector.
• (Released spectrum only) From the Filter list, select a different scan filter.
• In the Retention Time box, type a different retention time, and then click elsewhere
in the same row.
The corresponding scan number appears in the Scan Number box.
• In the Scan Number box, type a different scan number, and then click elsewhere in
the same row.
The retention time of the selected scan appears in the Retention Time box.
3. Click OK.
When you add multiple plots to a Spectrum view, the Spectrum view becomes a
MultiSpectrum view.

Creating a MultiSpectrum View and Changing Its Spectrum Plots

To compare spectrum plots using the same normalization level, you must use the
MultiSpectrum view (see Applying Local or Global Normalization in a MultiSpectrum View).

To specify the spectrum plots in a MultiSpectrum view, follow these procedures

• To change a Spectrum view to a MultiSpectrum view
• To add spectrum plots to a MultiSpectrum view
• To select a different spectrum for any of the plots in a MultiSpectrum view

Y To change a Spectrum view to a MultiSpectrum view

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Select a Spectrum view, click Spectrum, and choose Multi Spectrum from the dropdown
menu.
The view’s title bar changes to MultiSpectrum and a copy of the selected spectrum
appears below the original spectrum (Figure 73).

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Figure 73. MultiSpectrum view

Y To add spectrum plots to a MultiSpectrum view

1. Click the MultiSpectrum view to select it.

2. In the Workspace Options toolbar, click Spectrum and choose Multi Spectrum from the
dropdown menu.
A copy of the last plot appears at the bottom of the view.

Y To select a different spectrum for any of the plots in a MultiSpectrum view

• Use the Spectrum Ranges dialog box (see Selecting Spectra from the Spectrum
Ranges Dialog Box).
–or–
a. Click the plot to select it.
An active plot has a darker background than the other plots in the view (Figure 74).
Figure 74. MultiSpectrum view with plots from different raw data files

Selected
plot

b. In the Chromatogram view, select a point by using the pointer or the left and right
keyboard keys.

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Formatting a MultiSpectrum View

Formatting a MultiSpectrum View

For more information about the formatting options, see MultiSpectrum – Display Options
Toolbar.

Y To format the spectrum plots in a MultiSpectrum view

1. Click the MultiSpectrum view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. Do one or more of the following in the Format area:
• To change the color of a spectrum plot, select the plot, and then click the Color icon
and select a color.
• To change the orientation of the plots from stacked to overlaid, click Plot Options
and choose Overlay from the dropdown menu.
• To change the skew of the overlaid plots, use the Skew slider.
• To change the relative elevation of the overlaid plots, use the Elevation slider.
• To add a backdrop to the overlaid plots, click Draw Backdrop.

Figure 75 show a MultiSpectrum view with two spectrum plots with a 25% overlay, a 45°
skew, and a backdrop.
Figure 75. MultiSpectrum view with two overlaid spectrum plots

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Linking and Releasing Spectra to and from a Chromatogram

Linking and Releasing Spectra to and from a Chromatogram

When you add a Spectrum view to the Workspace, the application automatically links the
spectrum to the active chromatogram trace. You can release the spectrum from the trace and
link it to another trace.

See these topics:

• Linkage States for a Spectrum Plot
• Releasing a Linked Spectrum from the Chromatogram
• Linking a Spectrum View to a Chromatogram

Linkage States for a Spectrum Plot

Table 27 describes the linkage states for a spectrum plot.
Table 27. Linkage states for a spectrum plot
Linkage state Description
Linked to a When a spectrum plot is linked to a chromatogram, clicking another time
chromatogram point in the linked chromatogram changes the displayed spectrum.

The title bar of the Spectrum (or MultiSpectrum) view describes the linked
chromatogram by its view number and trace number in the following
format:
CxTy

where
x = the number of the Chromatogram view
y = the number of the trace in the Chromatogram view
Available for The following text is appended to the title bar of the Spectrum (or
linking MultiSpectrum) view—Select Trace to Link.

When a spectrum plot is available for linking, clicking any chromatogram

in the Workspace links the plot to the selected chromatogram, updates the
spectrum plot to the selected time point, and changes the appended text in
the view’s title bar to CxTy.
Released When you release a spectrum plot from its chromatogram, the text
appended to the Spectrum (or MultiSpectrum) view changes to Released.

In the Released state, the spectrum plot is not affected by any actions in a
Chromatogram view. To update a released spectrum plot, you must use the
Spectrum Ranges dialog box.

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Averaging Spectra

Releasing a Linked Spectrum from the Chromatogram

For a spectrum in a Spectrum view, the text appended to the view’s name indicates the linkage
status of the spectrum. For a MultiSpectrum view, the appended text applies to the selected
spectrum.

Y To release a spectrum from its linked chromatogram

Right-click the spectrum and choose Release from Chromatogram.

The application appends the following text to the view’s name: Released.

Note After you release a spectrum, you can use the Spectrum Ranges dialog box to
select a different detector, scan filter, or raw data file.

Linking a Spectrum View to a Chromatogram

Y To link a spectrum to a chromatogram

1. Right-click the spectrum and choose Link to Chromatogram.

2. Click the chromatogram to link it.
To describe the linkage state, the application appends the following text to the view’s
name:
CxTy
where x indicates the Chromatogram view’s number and y indicates the trace number

Note When a spectrum is linked to a chromatogram, you cannot use the Spectrum
Ranges dialog box to select a different detector, scan filter, or raw data file.

Averaging Spectra
You can increase mass accuracy and reduce noise in a spectrum by averaging the spectrum over
an appropriate scan range.

To generate an averaged spectrum, the application uses the original intensity versus frequency
data from the mass spectrometer, bins the data into frequency intervals, and then uses the
instrument’s mass calibration file to convert the frequency data to m/z values.

You can create an averaged spectrum by using the Tools area of the Chromatogram –
Workspace Processing toolbar or the Spectrum Ranges dialog box.

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See these topics:

• Creating an Average Spectrum from the Chromatogram Shortcut Menu
• Creating an Average Spectrum from the Spectrum Ranges Dialog Box

Creating an Average Spectrum from the Chromatogram Shortcut Menu

When a spectrum is linked to a chromatogram trace, you can use the Average Spectrum
shortcut menu command to create an average spectrum.

Y To create an average spectrum by using the shortcut menu

1. Right-click the Chromatogram view and choose Average Spectrum from the shortcut
menu.
The cursor changes to the averaging cursor: .
2. Drag the pointer through the time range that defines the scans to average.
A red line in the Chromatogram view marks the time range.
Figure 76 shows a Workspace page with two Spectrum views. The Spectrum view on the
right shows the average spectrum for the time range.

Tip To retain the average spectrum while working with the chromatogram, release the
spectrum from the chromatogram.

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Figure 76. Workspace with a Chromatogram view and two Spectrum views
Time range marker

Average spectrum

Creating an Average Spectrum from the Spectrum Ranges Dialog Box

Y To create an average spectrum by using the Spectrum Ranges dialog box

1. Right-click the Spectrum view of interest and choose Ranges.

The Spectrum Ranges dialog box opens.
2. In the Retention Time box, enter the time range for averaging spectra in the following
format:
RTstart–RTend
Figure 77. Spectrum Ranges dialog box with a time range for the averaged spectrum

3. Click Apply.

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Subtracting Background Spectra

Subtracting Background Spectra

The solvent or other noise can create unwanted background spectra. You can subtract them
from the spectrum for one or two ranges. The background subtraction algorithm subtracts an
average of the selected scans and redraws the spectrum. The spectrum view header shows the
number of subtracted scans. For example, SB: 12 indicates that the algorithm has applied
background subtraction (SB) to the spectrum by using 12 scans. To the right of that is
subtraction range_1 and range_2.

See these topics:

• Using the Tools Commands to Subtract Background Spectra
• Using the Spectrum Ranges Dialog Box to Subtract Background Spectra

Using the Tools Commands to Subtract Background Spectra

You can use the background subtraction commands in the Tools area of the Chromatogram –
Workspace Processing toolbar to subtract background spectra in a linked spectrum.

Y To subtract background spectra from the spectrum

1. Select the Chromatogram view of interest.

2. In the Tools area on the Workspace Processing toolbar, click the Subtract Background
(one range) icon, , or the Subtract Background (two ranges) icon, , to
background subtract over one or two ranges, respectively.
3. Drag the pointer through the time ranges that define the scans to subtract: once for one
range or twice for two ranges. Or, enter the time ranges in the Range1 and Range2 boxes.
You can undo the background subtraction by clicking the Subtract Background (one
range) icon, , or the Subtract Background (two ranges) icon, , again.
Figure 78 shows the process of dragging the pointer across range 1.
Figure 78. Background subtraction process for the first time range

Range 1 cursor

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Figure 79 shows the process of dragging the pointer across range 2.

Figure 79. Background subtraction process for range 2

Range 2 cursor

Figure 80 shows the selected background-subtraction ranges and the spectrum produced
by subtracting 24 scans.
Figure 80. Spectrum produced by subtracting 24 scans

Spectrum
produced by
subtracting
24 scans

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Exporting a Selected Spectrum to a New Raw Data File

Using the Spectrum Ranges Dialog Box to Subtract Background Spectra

When a Spectrum view is not linked to a chromatogram, use the Spectrum Ranges dialog box
to subtract background spectra.

Y To subtract background spectra by using the Spectrum Ranges dialog box

1. Select the Spectrum view of interest.

2. In the Spectrum – Workspace Options toolbar, click Spectrum and choose Ranges from
the dropdown menu.
The Spectrum Ranges dialog box opens.
3. Use the File Name list and the Retention Time or Scan Number box to define the
spectrum.
4. In the Subtract Background list, select 1 Range or 2 Ranges.
5. Enter the time range for the background spectra in the Background Range 1 and
Background Range 2 boxes as appropriate.
6. Click Apply.

Exporting a Selected Spectrum to a New Raw Data File

Raw data files can contain gigabytes of data. To work with only a selected spectrum, you can
export the spectrum to a new raw data file. The new raw data file contains the selected
spectrum and the sample data from the original raw data file and has a much smaller file size.

Y To export a spectrum to a new raw data file

1. Open a raw data file.

2. Set up the spectrum of interest in a Spectrum or MultiSpectrum view.
The spectrum can be a single scan, a portion of a scan, an averaged spectrum, or a
composite spectrum.
3. Click the spectrum plot of interest to select it.
4. Click the Workspace Options tab to open the Workspace Options toolbar.
5. Click Exports and choose Write to .RAW from the dropdown menu.
The Export Data dialog box opens. The File name box contains the original file name
appended with Scan[scan number].

Note When the raw data file contains Advanced Peak Determination (APD) data—
such as species ID, monoisotopic mass, and top peak in the cluster—the exported file
does not include the APD data.

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6. Browse to an appropriate storage folder and rename the file as necessary.

7. Click Save.
The new raw data file contains only the exported spectrum and the export date, which is
listed in the File Header report as the acquisition date and time.
The application records the creation of new raw data file in the Audit Trail.

Using Precursor Markers to Open Data-Dependent Scans

You can add these two markers to the tops of the spectrum peaks in precursor scans: Precursor
Flag and Nearby Precursors. Double-clicking these markers opens the data-dependent scans in
another view.

For information about turning on these markers and using them, see these topics:
• Using the Precursor Flag Marker
• Using the Nearby Precursors Marker

Using the Precursor Flag Marker

The application can label the spectrum peaks that triggered data-dependent scans with
triangles called precursor markers.

Y To turn on the precursor flag marker

1. Click a Spectrum view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Labels area, click the Precursor Flag icon, .
Figure 81 shows a full MS scan with a precursor flag at m/z 195.0877.

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Figure 81. Spectrum view with the Precursor Flag marker turned on
Precursor Flag label

Precursor marker in the

full MS1 scan

Y To display the fragmentation scan triggered by the precursor ion

Double-click a peak’s precursor marker to display its product ion spectrum in a separate
Spectrum view.
Figure 82 shows a full MS1 scan with one precursor ion at m/z 195.0877 and its
data-dependent product ion spectrum in a second Spectrum view.

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Figure 82. Precursor and product ion scans in separate Spectrum views

Precursor marker on the Precursor marker on the

m/z 195.0877 peak in the m/z 138.0662 peak
full MS1 scan
Precursor marker on the
m/z 110.0713 peak
Data-dependent MS2 scan for
m/z 195.0877

Figure 83 shows the product ion scans for the two marked precursor ions in the
data-dependent MS2 scan.

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Figure 83. Data-dependent MS2 scan with two precursor peaks and their MS3 product ion
scans

MS2 scan with two

precursor markers

Data-dependent
MS3 scan for
m/z 110.0713

Data-dependent
MS3 scan for
m/z 138.0662

Using the Nearby Precursors Marker

Use the Nearby Precursors marker to mark the peaks in the current MS(n-1) scan that the
instrument selected for data-dependent MSn acquisition in the current scan or another scan.
The application searches for matching data-dependent scans (by the precursor ion’s m/z value
within the specified mass tolerance) within the specified time range of the current scan.

Double-clicking a marker opens the average spectrum of the data-dependent scans in a

separate Spectrum view or individual spectrum plots for each data-dependent scan in a
MultiSpectrum view.

You can change the default behavior for this marker on the Workspace Options page.

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Y To specify the display options for the fragmentation scans

1. In the toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Workspace Options.

Tip Do not confuse this with the main Workspace Options toolbar.

3. In the Nearby Precursor area, specify the search and display options for the
data-dependent scans:
• Set the time range to search within the raw data file.

Tip Consider the experiment type when setting the time range. For infusion
experiments consider setting the search range to the entire file. For
chromatography experiments, consider basing the search range on the
chromatographic peak width.

• Set the maximum number of hits (matching data-dependent scans) to return.

• Set the plot type for the data-dependent scans to an average spectrum in a Spectrum
view or individual plots in a MultiSpectrum view.
• Set the mass tolerance for matching the m/z values of the peaks in the current scan to
the m/z values of the precursor ions for the data-dependent scans in the next level of
MS/MS activation.

Y To turn on the Nearby Precursor marker

1. Click a Spectrum view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Labels area, click the Precursor Flag icon, .

Y To display the related data-dependent scans for a precursor ion

Double-click the peak’s Nearby Precursor marker ( ).

Figure 84 shows a precursor scan with Nearby Precursor markers on the left and the average
spectrum for the related data-dependent scans on the right.

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Figure 84. Full MS scan with Nearby Precursor markers and an average spectrum of its four
related data-dependent scans (matching m/z value within specified RT)

Nearby Precursor marker for Average

m/z 346.1218 spectrum

Figure 85 shows the four matching (by precursor m/z value) data-dependent scans that the
application found within the specified retention time of scan #1795.
Figure 85. MultiSpectrum view showing the data-dependent scans

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Selecting Spectra from an MSn Tree

Selecting Spectra from an MSn Tree

On the MSn Browser page in the Info Bar view, you can select scans of interest from an MSn
spectrum tree. You can also select to display an average spectrum for all the MSn scans of a
specific precursor ion or a composite spectrum that includes the mass peaks from all the
fragmentation scans that originate from the same precursor ion. For information about the
parameter settings and the MSn tree structure, see MSn Browser Page.

Follow these procedures:

• To display the MSn Browser page
• To filter the MSn tree by a selected time range
• To filter the MSn tree by a selected mass range
• To modify the grouping of the scan data in the MSn tree
• To expand the entire MSn tree
• To display all the individual scan items
• To display an average spectrum
• To normalize a composite spectrum

Y To display the MSn Browser page

1. Open a raw data file (see Opening Raw Data Files or Sequence Files).
2. In the Info bar, click the MSn Browser tab.
In the MSn Parameters area, use the Time Range and Mass Range settings in the MSn
parameters area to filter the spectra in the MSn Tree area. Use the Mass Tolerance setting
to change the grouping of the MSn nodes.
In the MSn Tree area, the branches for each MS2 precursor appear as nested groups
ordered by their increasing m/z value. Expanding an MS2 precursor node displays the
MS3 precursor nodes when available, expanding the MS3 precursor nodes displays the
MS4 precursor nodes when available, and so on.
When an MSn level includes more than one scan, the application displays the average
spectrum for the level. When the experimental data includes multiple fragmentation
levels, for example MS2 and MS3, the MS3 level includes a composite spectrum made up
of the MS2 precursor scan and the MS3 fragmentation scans. An MS4 level node includes
a composite spectrum made up of the MS2 and MS3 precursor scans and the MS4
fragmentation scans.

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Y To filter the MSn tree by a selected time range

• In the MSn Parameters area, in the Time Range (min) box, type the time range that
you want to review.
–or–
a. In the MSn Parameters area, select the Track check box.
b. In the Chromatogram view, drag the pointer across the x-axis range that you want to
review.
The selected time range appears in the Time Range (min) box, and the MSn Tree view
displays the scans within the selected time range.

Y To filter the MSn tree by a selected mass range

In the Mass Range box, type a beginning mass and an ending mass, separated by a hyphen
(lowest mass-highest mass). The specified mass range must be within the mass range of the
scan data.
The MSn Tree view displays only the scans where the mass of the precursor ion falls
within the specified mass range.

Y To modify the grouping of the scan data in the MSn tree

In the Mass Tolerance box, type an m/z value from 0.00 to 10.00.
Figure 86 shows the effect of decreasing the mass tolerance from 0.50 to 0.00 and the
effect of increasing the mass tolerance from 0.50 to 1.00.
• Decreasing the mass tolerance below that of the experimental data increases the
number of MS2 precursor nodes.
• Increasing the mass tolerance from 0.50 to 1.00 combines the scans under the MS2
Precursor 100.08 node with the scans under the MS2 Precursor 101.06 node, as the
mass difference between these nodes is less than 1.00.

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Figure 86. Effect of changing the mass tolerance on the MSn tree
Decrease from 0.50 to 0.00 Increase from 0.50 to 1.00

Y To expand the entire MSn tree

Right-click the MSn Tree area and choose Expand List.

Y To display all the individual scan items

Right-click the MSn Tree area and choose Include Individual Scans.

Y To display the spectrum for a single scan

In the MSn Tree area, double-click any of the Single Spectrum Precursor items or any of
the Scan Number at Retention Time items.
A new Spectrum view appears in the Workspace.

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Y To display an average spectrum

In the MSn Tree area, double-click an Average Spectrum MSn precursor values (scan
range) item.
A new Spectrum view appears in the Workspace. The scan header includes the term
Average Spectrum, the number of averaged scans, and the scan number range for the
individual scans.
Figure 87 shows average MS2 level spectrum.
Figure 87. Average spectrum of two individual scans

Y To display a composite spectrum

Note The MSn tree includes a composite spectrum for each branch that includes
multiple fragmentation levels.

In the MSn Tree area, double-click a Composite Spectrum MSn precursor values item.
A new Spectrum view appears in the Workspace. The scan header includes the term
Composite Spectrum and the number of averaged scans.
Figure 88 shows a composite spectrum and the two scans (MS2 and MS3 fragmentation
levels) that the application combined to create it.

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Figure 88. Composite spectrum and individual MS2 and MS3 scans

Y To normalize a composite spectrum

Select the Normalize Composite Spectrum check box, and then double-click the
composite spectrum of interest in the MSn tree.
Figure 89 shows the normalized composite spectrum for scans 3760 and 3761.

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Specifying the Display Options for a Spectrum View

Figure 89. Normalized composite spectrum versus the individual MS2 and MS3 scans

Composite
spectrum

Scan# 3760, an
MS2 level scan

Scan# 3761, an
MS3 level scan

Specifying the Display Options for a Spectrum View

Use the Spectrum – Display Options toolbar to customize the display of the selected
Spectrum or MultiSpectrum view.

To specify the display options for the Spectrum view, follow the procedures in these topics:
• Modifying the Scan Header for the Spectrum View
• Applying Local or Global Normalization in a MultiSpectrum View
• Labeling Spectrum Peaks
• Labeling APD Data in Spectrum Peaks
• Labeling Mass Spectrum Peaks with Chemical Formulas
• Changing the Y-Axis Scale of a Spectrum View

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Specifying the Display Options for a Spectrum View

Modifying the Scan Header for the Spectrum View

By default, the scan header for the Spectrum view displays the following: Short File Name,
Scan Number, Retention Time, Average Number of Scans, Background Subtraction Scan
Numbers, Normalized Intensity, Polarity, Scan Filter String, and Mass Ranges.

Figure 90 shows a scan header for scan #166 at a retention time of 2.01 minutes and with
background subtraction applied to two ranges.
Figure 90. Default scan header for scan #166 in steroids04.raw with background subtraction
Short file name
Scan number (#)
Retention time (RT)
Number of scans averaged (AV)
Background subtraction (SB): number of
subtracted scans, range 1, range 2
Normalized intensity level (NL)

Scan Filter String

Y To add or delete information from the scan header for a Spectrum view

1. Open a raw data file.

2. If you are using a custom layout that does not include a Spectrum or MultiSpectrum view,
in the Workspace Options toolbar, click Spectrum or Multi Spectrum.
3. Click the Spectrum (or MultiSpectrum) view of interest.

Note The selections that you make in the Scan Header dialog box affect only the
selected Spectrum view (or MultiSpectrum view).

4. In the Spectrum – Display Options toolbar, click Scan Header.

The Scan Header dialog box opens (Scan Headers and Scan Header Abbreviations).
5. Do the following:
• To display a field, select its corresponding check box.
• To hide a field, clear its corresponding check box.
6. Click OK.

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Applying Local or Global Normalization in a MultiSpectrum View

In a MultiSpectrum view, you can normalize the mass spectra to the most intense peak in all
the spectra, or you can normalize the mass spectral peaks in each spectrum to the most intense
peak in the spectrum.

For information about displaying spectra in a MultiSpectrum view, see Creating a

MultiSpectrum View and Changing Its Spectrum Plots.

Y To normalize each spectrum separately

In the Normalization area of the Multi Spectrum – Display Options toolbar, click Local.
Figure 91 shows a set of locally normalized spectrum plots. Each spectrum is normalized
to the most intense peak in the spectrum.
Figure 91. Local normalization

Base peak in
scan# 3760,
NL=2E5

Base peak in
scan# 3761,
NL=1.6E1

Y To normalize the spectra against the most intense peak across the spectra

In the Normalization area of the Multi Spectrum – Display Options toolbar, click the
Global icon, .
Figure 92 shows the effect of global normalization where both spectrum plots are
normalized to the largest peak across the plots. The global normalization (GNL) is equal
to 2E5, and the low intensity peak in scan# 3716 is barely visible.

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Figure 92. Global normalization

Labeling Spectrum Peaks

Use the Labels area of the Spectrum – Display Options toolbar to add or remove labels that
appear above the spectrum peaks. By default, the application adds the mass-to-charge label to
each mass spectrum peak.

Note The Precursor Flag and Nearby Precursor icons do not add text labels to the
spectrum peaks; these icons add markers to the precursor scans. For instructions on how
to use these markers, see Using Precursor Markers to Open Data-Dependent Scans.

Y To add or remove the labels for the mass spectrum peaks

1. Open a raw data file and display a spectrum in a Spectrum view (or MultiSpectrum view).
2. Click the Spectrum (or MultiSpectrum) view of interest.
3. Click the Display Options tab to open the Display Options toolbar.
4. In the Labels area, click the labels that you want to display.

Note With the default value set to 0 in the Label Threshold box, the application
labels all the mass peaks.

5. (Optional) To avoid labeling low-intensity mass peaks, type a higher relative intensity
value in the Label Threshold box.
Figure 93 shows a mass spectrum with several labels and a labeling threshold of 50%
relative intensity.

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Figure 93. Mass spectrum with multiple labels

Intensity
threshold

Selected
labels

Peak
labels

Labeling APD Data in Spectrum Peaks

Use the Labels area of the Spectrum – Display Options toolbar to add or remove labels
specific to Advanced Peak Determination (APD) data—such as species ID, monoisotopic
mass, and top peak in the cluster.

Note To display the APD extended data in the FreeStyle application, you must write the
extended data to the raw data file during acquisition. You can write the APD extended
data to the raw data file for the Exploris and Tribrid MS instruments. Depending on the
instrument, you might need to set the development diagnostics menu to write the extra
raw data to the raw data file.

The APD algorithm improves the determination of the charge states and monoisotopic m/z
values of isotopic envelopes.

Monoisotopic mass—The monoisotopic mass of a species is the mass obtained using the exact
mass of the most abundant isotope of each constituent element.

Top peak—A centroid is labeled as the top peak of an isotope distribution by the APD
algorithm if it triggered a new charge state detection based on the surrounding peak pattern.

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Average mass—The average mass is the average of the neutral masses of the top peaks of the
charge envelope members. The average mass is available instead of the monoisotopic mass if
the species is not isotopically resolved. When the APD algorithm identifies the mass as an
average mass, the application does the following:
• Labels the mass spectral peaks with average mass in the active spectrum view.
• Labels the mass spectral peaks with the label AvgM in the format: AvgM=123.05.
• For the mass spectral peaks with no identified average mass, applies no label.

Y To add or remove the APD-specific labels

1. Open a raw data file that contains APD data, and display a spectrum in a Spectrum view
(or MultiSpectrum view).
2. Click the Spectrum (or MultiSpectrum) view to select it.
3. Click the Display Options tab to open the Display Options toolbar.
4. In the Labels area, click the labels that you want to display.
• Displays the monoisotopic mass and peak label (MM) in the Spectrum view.
Alternatively, when the species is not isotopically resolved, this icon displays the
average mass (AvgM) instead of the monoisotopic mass.
• Displays a species ID label (S) to the mass spectral peaks that belong to the same
chemical species (with the same species ID).
• Displays a label (T) on the top peak in the cluster as identified by the Advanced
Peak Determination algorithm.
Figure 94. APD labels

Identifies this peak

as top of cluster
Monoisotopic mass
Species ID

Labeling Mass Spectrum Peaks with Chemical Formulas

You can add formula labels to the peaks in your mass spectra.

Note When calculating the elemental compositions of all the mass peaks in a spectrum,
the application uses the settings on the default Elemental Composition page. When you
change the settings on this page, the new settings take effect after you restart the
application.

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Tip When determining the chemical formula for each mass peak, the application assumes
that the current mass peak is an A0 peak; that is, it assumes that the peak corresponds to a
monoisotopic ion, and the peaks at higher m/z values are part of the ion’s isotope pattern.
After determining the best matching formula for the current mass peak (by using the
accurate mass and isotope pattern), the application analyzes the next mass peak, again
assuming that the peak is an A0 peak.

Use the Isotope Simulation page to calculate the theoretical isotope patterns for the
displayed formulas.

Y To add formula labels to the mass peaks in a spectrum plot

1. Click a Spectrum view to select it.

2. Open the Workspace Processing toolbar.
Figure 95. Workspace Processing toolbar for the Spectrum view

Note (Optional) To ignore low-intensity peaks, type a higher intensity threshold in

the Display Options toolbar – Label Threshold box.

3. On the Elemental Composition page in the Info Bar, select the Pick Scan option, and
then click Calculate.
The Theoretical Mass, , and Delta Mass, , icons become available in the Labels area
of the Display Options toolbar.
Figure 96 shows the parameters on the Elemental Composition page and the chemical
formula labels in the Spectrum view. You can use the Elemental Composition page to find
the best matching formulas for a specific mass peak.

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Figure 96. Mass peaks with chemical formula labels in the Spectrum view

Experimental m/z value and calculated chemical

formula for the ion

4. (Optional) To label the peaks with the theoretical mass for the displayed formula and the
mass difference between the experimental m/z value and the theoretical mass, click Theo.
and Delta Mass, respectively.
Figure 97 shows the mass peaks labels, from top to bottom: the experimental m/z value,
the formula and calculated m/z value of the ion, and the mass difference between the
experimental m/z value and the calculated m/z value.
Figure 97. Mass peaks with formula, theoretical mass, and delta mass labels

Experimental m/z value, calculated chemical formula for the ion, calculated m/z value for the
formula, and mass difference between the experimental m/z value and the calculated m/z value

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Changing the Zoom Level in a Spectrum View or MultiSpectrum View

Changing the Y-Axis Scale of a Spectrum View

You can select the relative intensity scale or the absolute intensity scale for the y axis of a
spectrum plot.

With the factory default template, a Spectrum View opens with the y-axis scale set to relative
intensity and a y-axis label of Relative Abundance.

Y To change the y-axis scale from relative intensity to absolute intensity

1. Click a Spectrum view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. In the Y-Scale area of the Display Options toolbar, click Absolute.
In the Normalization area, the y-axis maximum changes from 100% to the normalization
(NL) value (or slightly less than the NL value) for the scan, and the y-axis label changes
from Relative Abundance to Intensity (Figure 98).
Figure 98. Spectrum plot of (absolute) Intensity versus m/z value

Changing the Zoom Level in a Spectrum View or MultiSpectrum View

You can zoom in on or out of a region of a spectrum by using the Spectrum – Zoom Options
toolbar, the MultiSpectrum – Zoom Options toolbar, or the mouse pointer.

Follow these procedures:

• To incrementally zoom in and out on the x axis
• To zoom in on a specific section of the x axis
• To incrementally zoom in and out on the y axis
• To reset the zoom level of the x and y axes

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Changing the Zoom Level in a Spectrum View or MultiSpectrum View

Y To incrementally zoom in and out on the x axis

1. Select the view.

2. To zoom in, click the Zoom In X icon, , in the Zoom Options toolbar.
Each click zooms in on a 50% smaller section of the x axis—that is, it decreases the
displayed range by a factor of 2.
3. To zoom out, click the Zoom Out X icon, , in the Zoom Options toolbar.
Each click increases the displayed m/z range by 200%.

Y To zoom in on a specific section of the x axis

Drag the pointer horizontally across the specific section of the x axis.

Y To incrementally zoom in and out on the y axis

1. Select the view.

2. In the Zoom Options toolbar, click the Zoom In Y icon, .
Each click zooms in on a 50% smaller section of the y axis. For example, in a Spectrum
view where the y axis is set to relative abundance, clicking once zooms in on the 0 to 50%
range, clicking twice zooms in on the 0 to 25% range, and so on.
3. To incrementally undo the zoom level, do the following:
• Click the Zoom Out Y icon, .
–or–
• Right-click the view and choose Undo Zoom.

Y To reset the zoom level of the x and y axes

1. Select the view.

2. Do either of the following:
• In the Zoom Options toolbar, click the Reset icon, .
–or–
• Right-click the view and choose Reset Scaling.

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Spectra-specific Toolbars

Spectra-specific Toolbars
Use the spectra-specific toolbars as follows:
• Spectrum – Workspace Processing Toolbar: For determining the elemental composition
of an ion; simulating isotope distributions; Xtract deconvolution; NIST library, mzVault
library, and mzCloud.org searches.
• Spectrum – Display Options Toolbar: For customizing the Spectrum view
• MultiSpectrum – Display Options Toolbar: For customizing the MultiSpectrum view
• Spectrum List – Display Options Toolbar: For formatting the spectrum list

These two toolbars are specific to the Spectrum, MultiSpectrum, and Chromatogram views:
• Zoom Options Toolbar: For adjusting the display of the spectra (and chromatograms).
• Text and Graphic Annotation Toolbar: For annotating spectra (and chromatograms) with
text, lines, boxes, and symbols.

Spectrum – Workspace Processing Toolbar

Use the features in the Elemental Analysis, Protein Analysis, and Library Search areas of the
Workspace Processing toolbar to analyze spectra.

Y To display the Workspace Processing toolbar for a spectrum

1. Click a Spectrum view or a MultiSpectrum view to select it.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.

Figure 99 shows the Workspace Processing toolbar, and Table 28 describes the toolbar
commands.
Figure 99. Workspace Processing toolbar

Table 28. Spectra-specific Workspace Processing commands (Sheet 1 of 2)

Command Description
Elemental Analysis area
Isotope Simulation Displays the Isotope Simulation page in the Info Bar, where you create a simulated
isotopic distribution spectrum of a chemical formula.

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Table 28. Spectra-specific Workspace Processing commands (Sheet 2 of 2)

Command Description
Protein Analysis area
Peptide Fragments Displays the Peptide Fragments page of the Info Bar, where you set parameters to
annotate the active spectrum using the CID or ETD activation types. Not available for
Multi Spectrum view.
Xtract Deconvolution Displays the Xtract page of the Info Bar, where you set parameters for the Xtract
algorithm. See Chapter 13, “Running the Xtract Algorithm on Spectra and
Chromatogram Data.”
Library Search area
Library Search menu
NIST Search Opens the Library Search page in the Info Bar, where you run a NIST library search on
the selected spectrum and displays the results in the NIST Search Results view. You can
modify the library search parameters on the Modifying a NIST Search from the NIST
Search Page of the Info Bar. See Performing a Local NIST or mzVault Library Search.
mzVault Search Opens the mzVault Search page in the Info Bar, runs an mzVault library search on the
selected spectrum, and displays the results in the mzVault Search Results and mzVault
Chemical Structure views.
mzCloud Search Uploads a spectrum to mzCloud.org for a search. The website opens and displays the
search parameters. Select the appropriate settings and click OK. The website displays
the results. See Searching the Online mzCloud Mass Spectral Database.
Export to NIST Exports a spectrum to the NIST application for a search. The NIST application opens
and displays the search results. See Exporting a Mass Spectrum to the NIST MS Search
Application.
Library Manager Opens the Thermo Library Manager dialog box, where you select or create NIST
libraries. See Managing Libraries.

Spectrum – Display Options Toolbar

Use the features in the Spectrum – Display Options toolbar to customize the Spectrum view
(see Specifying the Display Options for a Spectrum View). You can label the spectral peaks,
determine the elemental composition for a peak, and specify how the application normalizes
the spectrum.

Y To display the Spectrum – Display Options toolbar

1. Click a Spectrum view to select it.

2. Click the Display Options tab to open the Display Options toolbar.

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Figure 100 shows the Spectrum – Display Options toolbar. Table 29 describes the features in
the Spectrum – Display Options toolbar.
Figure 100. Spectrum – Display Options toolbar

Table 29. Spectrum – Display Options toolbar commands (Sheet 1 of 4)

Command Description
Format area
Color Displays the color palette, where you select the color of the spectrum.

Labels area

Use the features in the Labels area to annotate the mass spectrum.
Note The following features are unavailable when the raw data file does not include the requested information: Z+
Charge State, Peak Resolution, Noise, Baseline, Width, and S/N Signal To Noise.
Nearby Precursors Adds markers ( ) to the tops of the spectrum peaks in scans that have related
data-dependent scans. Depending on the configuration setting for the Nearby
Precursor Plot Type parameter, clicking a peak’s marker opens a Spectrum view with an
average spectrum for all the related data-dependent scans or a MultiSpectrum view with
a separate plot for each related data-dependent scan (within the specified RT window).
Mass to Charge Adds a mass-to-charge ratio label to each peak in the spectrum.
Z+ Charge State Adds a charge state label to each peak in the spectrum.
Peak Resolution Adds a peak resolution label to each peak in the spectrum.
Precursor Flag Adds one or more pink triangles to a peak if it triggered data-dependent scans.
Noise Adds a noise magnitude label to each peak in the spectrum.
Signal To Noise Adds a signal-to-noise label (intensity/noise) to each peak in the spectrum.
Width (m/r) Adds a width label to each peak in the spectrum.The width equals the mass, in mmu
units, divided by the peak resolution.
Baseline Adds a baseline label to each peak in the spectrum.

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Table 29. Spectrum – Display Options toolbar commands (Sheet 2 of 4)

Command Description
Flags Displays one of the following letters or symbols above flagged peaks:
• S—Saturated peaks are peaks with a signal too large to measure—that is, the signal
was so high that it was outside the dynamic range of the detector, causing
saturation.
• R—Reference peaks are peaks from a reference compound used for an internal
recalibration of a scan.
• L—Lock peaks are local references used to calculate the accurate mass of nearby
peaks.
• E—Exception peaks are peaks from a reference compound that are not used for
recalibration. These are typically small isotopes or fragments of the main
references.
• #—Mathematically modified peaks are peaks where the peak mass was recalculated
by the instrument, usually due to a calibration process.
• M—Merged peaks are peaks where the centroider combined two nearby peaks.
• F—Fragmented peaks are peaks separated into multiple peaks by the centroiding
activity.
• T—Top peak in the cluster identified by the Advanced Peak Determination
algorithm.
• U—Peak identified as an unresolved isotope where the resolution settings used to
acquire the data are not sufficient to resolve isotopes of a given mass and charge
state as determined from charge state deconvolution.
Monoisotopic Mass Displays the monoisotopic mass and peak label (MM) in the Spectrum view.
• For peaks in the cluster that are not monoisotopic, the label is shown as MM:1234.
• For the monoisotopic peak in the cluster, the label is shown as MM=1234.
Species ID Displays a species ID label (S) in the Spectrum view on the top of mass spectral peaks
that belong to the same chemical species (with the same species ID). For mass spectral
peaks that do not have assigned species ID, the application does not label the peak.

The application updates the Spectrum List view with a Species ID column and data.

Format: S=123
Elemental Comp. Displays the formula labels on all the peaks in the spectrum.

Theo. Labels the peaks in the mass spectrum with the theoretical mass-to-charge ratios of the
best matched ions.

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Table 29. Spectrum – Display Options toolbar commands (Sheet 3 of 4)

Command Description
Delta Mass Labels the peaks in the mass spectrum with the differences between the experimentally
determined mass-to-charge ratios and the theoretical mass-to-charge ratios.

The unit options are amu (atomic mass units), mmu (millimass units), and ppm
(parts-per-million).
Label Threshold Sets the percentage of the base peak so that the application labels only the peaks above
that percentage. For example, if the base peak is 100 percent and the label threshold
setting is 50 percent, the application labels only the peaks in the Spectrum view or
MultiSpectrum view that are at or above 50 percent.

Range: 0–100; default: 0%

Reference area
Displays or hides the reference and exception peaks in the Spectrum view.

Normalization area

Use the features in the Normalization area to specify how the application normalizes the mass spectrum.
Local Scales the y-axis range as a percentage.

Global Normalizes the spectra traces so that the most intense peak of all the spectra is 100
percent.

Available for a MultiSpectrum view.

Off Scales all spectra traces by using the maximum and minimum absolute values.
Note When you set the normalization to Off, the y-axis scale to Absolute, and
change the trace type, the new trace might not display. To remedy this, set the
normalization to Local and then to Off.
Min. Displays the minimum of the y axis. Enter a value in the box to change the minimum.
For local normalization, the value is a percentage. For normalization set to Off, the
value is an intensity.
Max. Displays the maximum of the y axis. Enter a value in the box to change the maximum.
For local normalization, the value is a percentage. For normalization set to Off, the
value is an intensity.
Y-Scale area

Use the features in the Y-Scale area to specify how the application scales the y axis.
Relative Plots the spectrum with the intensity expressed in counts.

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Table 29. Spectrum – Display Options toolbar commands (Sheet 4 of 4)

Command Description
Absolute Scales the spectrum so that the intensity of the most intense peak in the spectrum is
100 percent.

Y Axis

Use the features in the Y Axis area to specify how the application labels the y axis.
Show Labels Shows or hides the y-axis label.
Offset Axis Sets the location of the displayed plot a specified distance from the y axis.

The y-axis offset moves the x axis slightly to the right of the y axis so that you can see
plot details at low x-axis values.
X Axis area

Use the features in the X Axis area to specify how the application labels the x axis.
Show Labels Shows or hides the x-axis label.
Offset Axis Sets the location for the displayed plot a specified distance from the x axis.

The x-axis offset moves the y axis up slightly so that you can see plot details at low y-axis
values.
Legend area
Scan Header Opens the Scan Header dialog box, where you specify what scan header information
the Spectrum view displays. For additional information, see Scan Headers and Scan
Header Abbreviations.

MultiSpectrum – Display Options Toolbar

Use the features in the Spectrum – Display Options toolbar to customize the MultiSpectrum
view.
Figure 101. MultiSpectrum – Display Options toolbar

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In addition to the Display Options in the Spectrum view, Table 30 describes the Format and
Label options specific to the MultiSpectrum – Display Options toolbar. For information
about the other toolbar areas, see Spectrum – Display Options Toolbar.
Table 30. Spectrum – Display Options toolbar features
Commands Description
Format area
Plot Options menu
Stack Vertically stacks the spectrum traces.
Overlay Vertically overlays the spectrum traces with an optional horizontal
skew (time offset).
Draw Backdrop Draws a backdrop for overlaid plots.
Sets the skew angle (time offset) to a value from 0–45 degrees for an
overlay arrangement of spectrum traces.

To set the skew, drag the Skew slider.

Sets the vertical spacing for an overlay arrangement of spectrum
traces.

To set the vertical spacing, drag the Elevation slider. Move the
Elevation slider to the farthest left to overlay the plots on top of each
other.

Spectra-specific Views
The FreeStyle window has these spectra-specific views:
• Spectrum view: Displays a spectrum corresponding to the retention time or scan number
that you select in the Chromatogram view.
• MultiSpectrum view: Displays multiple spectra, but only the active spectrum updates
corresponding to the retention time or scan number that you select in the Chromatogram
view.
• Spectrum List view: Lists in tabular form the positions, intensities, and relative intensities
of the peaks in the Spectrum view.

For information about the shortcut menu for the Spectrum and MultiSpectrum views, see
Table 32.

For more information, see these topics:

• Spectrum View
• MultiSpectrum View
• Spectrum List View (in the Viewing Experiment and Instrument Information chapter)

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Spectrum View
The Spectrum view (Figure 102)displays a spectrum for the retention time that you either
select in a chromatogram trace or specify in the Scan Ranges dialog box.

For information about displaying and reviewing the spectral data in a raw data file, see the list
of topics at the beginning of this chapter, “Reviewing Spectral Data.”

Y To add a Spectrum view to the Workspace

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Spectrum and choose Insert View from the dropdown menu.

Figure 102 shows an example of a selected Spectrum view. The view’s title bar displays the
scan number of the displayed spectrum, the file name of the raw data file, and the view’s link
status. C1T1 specifies that the view is linked to trace 1 in Chromatogram view 1.
Figure 102. Spectrum view
Linkage status

Scan
header

Note You can set a minimum trace height value in centimeters on the Default Workspace
Options page. When you adjust the height of the Spectrum view, if its height becomes
smaller than the set minimum value, a scrollbar automatically appears to the right of the
view.

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The scan header describes the experiment. Table 31 describes the default scan header
information. If the scan header is not visible, resize the Spectrum view.
Table 31. Default header information symbols
Symbol Description
#S Scan number
RT Retention time
AV Averaged (followed by the number of averaged scans)
SB Subtracted (followed by the number of subtracted scans and the
scan range)
NL Normalization level
T Scan filter string

Right-clicking a Spectrum or MultiSpectrum view displays a shortcut menu (Figure 103) with
the commands described in Table 32.
Figure 103. Shortcut menu for the Spectrum view (left) or the MultiSpectrum view (right)

Table 32. Spectrum (or MultiSpectrum) view shortcut menu commands (Sheet 1 of 2)
Command Description
Reset Scaling Resets the scaling of the plot in the Spectrum view or all
the plots in the MultiSpectrum view.
Copy To Clipboard Copies an image of the Spectrum view to the Clipboard.
Undo Zoom Undoes the last zoom.
Redo Zoom Reapplies the last zoom that you undid.
Link to Chromatogram / Links or releases the selected spectrum from its
Release from Chromatogram chromatogram.
Ranges Opens the Spectrum Ranges dialog box for defining the
spectrum.

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Table 32. Spectrum (or MultiSpectrum) view shortcut menu commands (Sheet 2 of 2)
Command Description
Clear Structure Annotations Clears the structure annotations from the view and the
structure list from the Structures Annotation dialog box.
Undo Delete Spectrum Undoes the action of deleting a spectrum in a
MultiSpectrum view.
(MultiSpectrum view only)

These toolbars are available when a Spectrum view or MultiSpectrum view is selected:
• Spectrum – Workspace Processing Toolbar
• Spectrum – Display Options Toolbar
• Zoom Options Toolbar
• Text and Graphic Annotation Toolbar

MultiSpectrum View
Use a MultiSpectrum view to display multiple spectrum traces when you want to compare the
traces using the same normalization level or you want to simultaneously zoom in on the same
mass range.

Note In the Normalization area of the Spectrum – Display Options toolbar, the Global
icon is unavailable when the spectra being compared are in two separate views.

Instead of zooming in on each spectrum individually in a Spectrum view, use a

MultiSpectrum view to zoom in and out on all of the spectra at once. In a MultiSpectrum
view, the selected spectrum (the spectrum with the darker gray background) is the only one
that updates when you click the chromatogram.

You can perform background subtraction, Xtract deconvolving and deisotoping, isotope
simulation, or library searches on any selected spectrum in this view.

The MultiSpectrum view’s shortcut menu contains the same commands as that for the
Spectrum view with the exception of one additional command—Undo Delete Spectrum.

To create and work with a MultiSpectrum view, see Creating a MultiSpectrum View and
Changing Its Spectrum Plots.

For more information about the MultiSpectrum view and its shortcut menu, see Spectrum
View.

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Spectrum Ranges Dialog Box

Spectrum Ranges Dialog Box

Use the Spectrum Ranges dialog box to select the spectra to display, create an average
spectrum, and perform background subtraction.

Table 33 describes the columns in the Spectrum Ranges dialog box.

Table 33. Spectrum Ranges dialog box
Column Description
Display Specifies whether to add the defined spectrum to the Spectrum or
MultiSpectrum view.
Release Specifies the linkage status of the defined spectrum.

Selected: Releases the spectrum from the chromatogram.

Clear: Links the spectrum to the chromatogram.

File Name Specifies the location and file name of the raw data file with the
spectral data of interest.

When the spectrum is not linked to a chromatogram, a browse icon

is available for browsing to and selecting a different raw data file.
Detector Type Specifies the detector used to acquire the data.

Unavailable when the spectrum is linked to a chromatogram.

Filter Specifies the scan filter (same selections as the Scan Filters page of
the Info bar).

Unavailable when the spectrum is linked to a chromatogram.

Retention Time Specifies the retention time for a single spectrum or the retention
time range for an average spectrum.

The Retention Time and Scan Number columns are interactive;

that is, changing the retention time range updates the scan number
range.
Scan Number Specifies the scan number for a single spectrum or the scan number
range for an average spectrum.
Subtract Background Specifies whether to perform background subtraction or whether to
subtract the scans from one or two time ranges.
Background Range 1 Specifies the time range for one set of background scans.

Format: TimeStart–TimeEnd, where the start time point is earlier

than the end time point
Background Range 2 Specifies the time range for a second set of background scans.

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MSn Browser Page

MSn Browser Page

Use the MSn Browser page in the Info Bar to select spectra of interest from an MS tree view of
the spectra in a raw data file.

For information about displaying spectra, see Selecting Spectra from an MSn Tree.

Figure 104 shows the MSn Browser page, and Table 34 describes the parameters on the page.
Figure 104. Info Bar – MSn Browser page (with a collapsed MSn tree)

Note The MSn browser information page is not available for all Thermo Scientific mass
spectrometers.

Table 34. MSn Browser page parameters (Sheet 1 of 3)

Parameter Description
MSn Parameters
Time Range (min) Specifies the time range of the MSn Tree (see To filter the MSn
tree by a selected time range).

Limits the spectra available for display in the MSn tree to MS2 or
higher level scans acquired during the specified time range.

There are two ways to change the Time Range:

• Type the time range in minutes in the Time Range box.
The format is From-To.
• Select the Track check box, and then drag the pointer
horizontally across the Chromatogram view from the
minimum time to the maximum time of interest.

Default: 0.00–acquisition time (in minutes)

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Table 34. MSn Browser page parameters (Sheet 2 of 3)

Parameter Description
Track Activates the selection of a time range by using the mouse pointer.
To reset the time range, right-click the Chromatogram view and
choose Reset Scaling.

Default: Clear
Mass Range Specifies the mass range of the MSn Tree (see To filter the MSn
tree by a selected mass range).

The format is From–To.

Default: * (Mass range from the instrument method)

Mass Tolerance Specifies the mass tolerance for grouping the precursor nodes and
their associated scans. Decreasing the mass tolerance to 0.00
creates an MSn Tree where most, if not all, the individual scans are
associated with a separate MS2 precursor node (see To modify the
grouping of the scan data in the MSn tree).

Default: 0.05 m/z; Range: 0.00–10.00 m/z

Normalize Composite Normalizes the spectral peaks in a composite spectrum so that you
Spectrum can view low-intensity peaks from the higher-order fragmentation
spectra (see To normalize a composite spectrum).

Each MSn spectrum is individually normalized (NL) so that its

highest peak is displayed at a Relative Abundance of 100%;
therefore, the relative peak heights of this display are not
meaningful. For example, a composite spectrum for an MS3
experiment displays both the MS2 base peak and the MS3 base
peak at a Relative Abundance of 100% (unless the base peaks in
the MS2 and MS3 are within the specified mass tolerance) and
maintains all other relative abundances of the other ions in each
spectrum. When the base peaks in the MS2 and MS3 scans are
within the specified mass tolerance, the application averages these
peaks, causing the Relative Abundance of the MS3 base peak to be
less than 100%.
MSn Tree
MS2 Precursor nodes Display the precursor masses that triggered the MS2 level scans.
To display the spectrum tree for each node, click the expand icon
to the left of the node, or right-click the MSn Tree view and
choose Expand List.
MSn Precursor nodes Display the precursor masses that triggered the MSn level scans.

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MSn Browser Page

Table 34. MSn Browser page parameters (Sheet 3 of 3)

Parameter Description
Average Spectrum Double-click to display a Spectrum view with the averaged
spectrum for the selected MSn level. The MSn tree includes an
average spectrum when the data file includes scans for the selected
scan filter.
Composite Spectrum Double-click to display a Spectrum view with the composite
spectrum for all the MSn levels down to the selected MSn level.

A composite spectrum for an MSn level is a summed spectrum of

all the fragmentation scans derived from the same original
precursor mass down to the current level.
Single Spectrum or Double-click to display a Spectrum view for the specified scan
Scan Number at Time number.
Shortcut menu
Include Individual Displays the individual scans (see To display all the individual scan
Scans items).
Normalize Composite Normalizes the composite spectra (see To normalize a composite
Spectrum spectrum).
Expand List Expands all the MSn Precursor nodes (see To expand the entire
MSn tree).

Available when the MSn tree is collapsed.

Collapse List Collapses all the MS2 Precursor nodes.

Available when the MSn tree is expanded.

Export Copies the MSn Tree to the Clipboard.
Print Opens the Print dialog box where you can specify your print
preferences and print the contents of the MSn Tree.

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Reviewing Map Data

Use the Map View-specific toolbars and display options of the FreeStyle application to review
the map view data from mass spectrometer.

These procedures describe how to set the ranges and display options for a map view.

Contents
• Working with the Map View
• Adding Map Views to the Workspace
• Changing the Zoom level of a Map View
• Linking and Releasing Map to and from a Chromatogram
• Map View – Specific Toolbars
• Map Ranges Dialog Box
• Setting the Map Display Options
• Setting the Format Options

Working with the Map View

A map is a 2D or 3D representation of an analysis showing the mass scans acquired during an
analysis. Use the Freestyle application to open a raw data file, create map views, and customize
using the display options.

The Map View consists of a time point (x axis) versus an m/z value (z axis) versus a relative
abundance value (y axis) map plot. The scan header contains File name, RT range, Mass
range, NL values, and filter details. These values change based on the changes in the Map
View.

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Adding Map Views to the Workspace

Figure 105. Map View example

Adding Map Views to the Workspace

You can add multiple Map Views to the workspace.

Y To add a Map View

1. Open a raw data file (see Opening Raw Data Files or Sequence Files).
2. With the factory default layout, the Chromatogram view appears at the top and the
Spectrum view appears at the bottom.
3. In the Workspace Options toolbar, click Map View and choose Insert View from the
dropdown menu.
A new Map View opens for each selected chromatogram with RT on the x axis and m/z
on the z axis.

Note When the Workspace includes one or more Chromatogram views, a map view
of the selected or active Chromatogram view appears.

When the Workspace does not include a Chromatogram view, a map view of the
chromatogram associated with the active spectrum view appears.

You can create multiple map views, but a single map view cannot have multiple traces. To
increase the intensity, use the Up and Down arrow keys on your computer.

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Linking and Releasing Map to and from a Chromatogram

Displaying the Scan for a Time Point in Map View

Y To display the scan corresponding to a specific time point

1. Select the Map View.

2. Click any time point.
In the Map View, a red vertical marker, , indicates the selected data point. The Spectrum
view displays the spectrum for that retention time or scan number. The Retention Time
marker position in the chromatogram also is updated correspondingly to sync with the
Map View.

Linking and Releasing Map to and from a Chromatogram

When you create a Map View in a workspace, the application automatically links the map
view to the active chromatogram trace. The displayed map view changes when you click
another trace in the linked chromatogram view. If you do not want to change the map view,
you can release the map view from the trace and link to another chromatogram trace.

See these topics:

• Linking Status of a Map View
• Releasing a Linked Map View from the Chromatogram
• Linking a Map View to a Chromatogram

Linking Status of a Map View

When you link or release the map view, the application appends the linking status to the
view’s name.
Figure 106. Map View Linking Status
Linking State

Table 35 describes the different linking status for a Map View.

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Linking and Releasing Map to and from a Chromatogram

Table 35. Map View Linking Status

Linking status Description
Link to chromatogram When a Map View is linked to a chromatogram, the title bar of
the Map View displays the linked chromatogram by its view
number and trace number in the following format:

CxTy

where

x = the number of the Chromatogram view

y = the number of the trace in the Chromatogram view

Available for linking The following text is appended to the title bar of the Map
View—Select Trace to Link.

When a map view is available for linking, clicking any

chromatogram in the Workspace links the view to the selected
chromatogram, updates the RT marker in Map View and
displays the spectrum based on the selected time point in the
Chromatogram trace, and changes the appended text in the
view’s title bar to CxTy.
Released When you release a Map View from its chromatogram, the text
appended to the Map View changes to Released. In the
Released state, the Map View is not affected by any actions in
the Chromatogram view. The Spectrum view also does not
change as you navigate to different retention times. To update a
released Map View, you must use the Map Ranges dialog box.

Releasing a Linked Map View from the Chromatogram

In a Map View, the title bar of the view’s name indicates the linking status of the Map View.

Y To release a Map View from its linked chromatogram

Right-click the Map View and choose Release from Chromatogram.

The application releases the link to the chromatogram trace and appends the following
text to the view’s name: Released.

Note When you close the chromatogram trace linked to a Map View, the application
automatically releases the linking status. The Map View displays the RT marker,
which is not synced with a chromatogram or spectra.

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Changing the Zoom level of a Map View

Linking a Map View to a Chromatogram

Y To link a map to a chromatogram

1. Right-click the map and choose Link to Chromatogram.

The application appends the following text to the view’s name: Select trace to link.
2. Click the chromatogram trace to link.
The application links to the chromatogram trace and appends the following text to view’s
name:
CxTy
where x indicates the Chromatogram view’s number and y indicates the trace number.

Changing the Zoom level of a Map View

You can reset, zoom in, or out of a Map View using the mouse pointer, Zoom options toolbar,
or right-click menu options.
To understand more about zooming a map view, follow these procedures:
• Using the Mouse Pointer
• Using the Zoom Options Toolbar

Using the Mouse Pointer

Y To zoom in and out using the mouse pointer

Select the view, and do any of the following:

• To zoom in the retention time (x scale), position the mouse pointer and drag horizontally
across the specific section. Release the mouse to zoom.
• To zoom in the m/z scale, position the mouse pointer and drag vertically across the
specific section. Release the mouse to zoom.
• To zoom in both x and m/z scales, click and drag the mouse within the map to form a box
containing the region to zoom. Release the mouse to zoom.
• To return to the previous scale, right-click and select Undo Zoom.

Using the Zoom Options Toolbar

Y To zoom in and out on the x axis

1. Select the view.

2. To zoom in, from the Zoom Options toolbar, click Zoom In X.
Each click zooms in the x axis larger by a factor of two (2) to show more detail. For
example, you can change the x-axis range from 0–20 to 5–15.

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Map View – Specific Toolbars

3. To zoom out, click Zoom Out X.

Each click increases the Time (min) range from the center. For example, you can change
the x-axis range from 7.5–12.5 to 5–15.

Y To zoom in and out on the y axis

1. Select the view.

2. To zoom in, from the Zoom Options toolbar, click Zoom In Y.
Each click zooms in on the y axis. by a factor of two from the current baseline.
3. To zoom out, click Zoom Out Y.
Each click zooms out on the y axis by a factor of two. For example, you can change the
y-axis range from 0–25 to 0–50.

Y To reset, undo and redo zoom of the x and y axes

1. To reset scaling, do the following:

• Select the view and from the Zoom Options toolbar, click Reset.
–or–
• Right-click the view and choose Reset Scaling.
2. To undo or redo zooming, right-click the view and choose Undo Zoom or Redo Zoom
respectively.

Map View – Specific Toolbars

Use the following map view-specific toolbars to create, format, and set map ranges and display
options of map views.
• Map View – Workspace Options Toolbar
• Map View – Display Options Toolbar

Map View – Workspace Options Toolbar

Use the features in the Workspace area of the Workspace Options toolbar to create map views
and set the map ranges.

Y To display the Map View-specific Workspace Options toolbar

1. Click the chromatogram or spectrum to select it.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Map View and choose Insert View from the dropdown menu.
The Map View-specific workspace options appear.

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Map View – Specific Toolbars

Figure 107. Map View – Workspace Options toolbar

Table 36. Workspace Options – Workspace area features for the Map View
Command Description
Map View menu
Ranges Opens the Map Ranges dialog box to specify a filename and scan
filter for the map.
Insert View Adds a Map View of the active chromatogram or spectrum.

Displays a map of the currently selected raw file in the map view
where you can view the retention time (RT) and m/z values.

Map View – Display Options Toolbar

Use the features in the Display Options toolbar to customize the Map View. You can format
the 3D or 2D representation of the map view, define the colors based on intensity, label the
axes, and specify the band width.

Y To display the Map View – Display Options toolbar

1. Click a Map View to select it.

2. Click the Display Options tab to open the Display Options toolbar.
The Map View specific display options appear.
Figure 108. Map View – Display Options toolbar

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Map View – Specific Toolbars

Table 37. Map View – Display Options toolbar icons (Sheet 1 of 2)

Icon Description
Format area

Use the parameters in the Format area to represent the Map View in 3D or 2D.
Plot Options menu
Density Displays different shades in the 2D representation of the map view, where colors represent
the m/z bands categorized based on the intensity percentage.
Overlay Displays the 3D representation of the map view.
Sets the skew angle (time offset) to a value from 0–45 degrees for an overlay arrangement of
Map View.
Sets the vertical spacing for an overlay arrangement of Map View.

Adds a backdrop to overlaid views.

Sets the filling style for map view. This is applicable only for 3D representation of the map
view (when Overlay is selected).

Color area

Displays the color palette, where you select the colors of the traces in the Map View.
Shade
Line Sets the color of the framing lines
Fill Solid Sets the color of the solid fill.
Backdrop Changes the color of the backdrop
Gray Scale Plots the map in gray scale
Log Scale Displays and clears the color of the map in a logarithmic scale.
Factor width value for the log scale.

Range: 1.1 through 20.0

Smoothing Displays a smoothened density plot for the color bands (bins of m/z values) in the vertical
direction. This is applicable only for 2D representation of the map view.

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Map View – Specific Toolbars

Table 37. Map View – Display Options toolbar icons (Sheet 2 of 2)

Icon Description
X Axis area

Use the parameters in the X Axis area to specify how the application labels and displays the x axis.
Show Labels Shows or hides the x-axis label.
(X Axis)
Offset Axis Sets the location of the displayed plot at a specified distance from the x axis.
(X Axis)
Y Axis area

Use the parameters in the Y Axis area to specify how the application labels the y axis.
Show Labels Shows or hides the y-axis label.
(Y Axis)
Offset Axis Sets the location of the displayed plot a specified distance from the y axis.
(Y Axis)
Z Axis area

Use the parameter in the Z Axis area to specify how the application labels the z axis.
Show Labels Shows or hides the z-axis label.

Axis Options area

Use the parameter in the Axis Option area to create grid lines in the map view.
Show Grid Displays or clears the grid lines.
Lines

Band Width area

Use the parameter to specify the band width in amu units.

Specifies a band width value from 0.001 to 50.

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Map Ranges Dialog Box

Map-Specific Views
The Map View displays a 2D or 3D representation of the map view of the selected
chromatogram or spectrum. The title bar of the view displays the file name of the raw data
file, RT range, Mass range, NL values, and the view’s link status. C1T1 specifies that the view
is linked to Trace 1 in the Chromatogram1 view.
Figure 109. Map View Title bar

Map Ranges Dialog Box

Use the Map Ranges dialog box to select the raw data file to display in the Map View, detector
type used to acquire the data, scan filter, and time and mass ranges.

Y To open the Map Ranges Dialog box

In the Workspace Options toolbar, click Map View and choose Ranges from the dropdown
menu.
Figure 110. Map Ranges Box

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Map Ranges Dialog Box

Table 38 describes the columns in the Map Ranges dialog box.

Table 38. Map Ranges dialog box
Column Description
Release Specifies the linking status of the Map View.

Selected: Releases the map view from the chromatogram. Lets you to
change the Filter name and raw file.

Clear: Links the map view to the chromatogram. You cannot change the
raw file and filter name.
File Name Specifies the location and file name of the raw data file.

When the map view is not linked to a chromatogram, a browse icon is

available to select a different raw data file.
Detector Type Specifies the detector used to acquire the data. By default, the detector type
is set to MS and is non-editable.

Unavailable when the map view is linked to a chromatogram.

Time Specifies a valid time range based on the raw file settings. Default: ‘*’.
• where ‘*’ indicates All.
Mass Specifies a valid mass range based on the raw file settings. Default: ‘*’.
• where ‘*’ indicates All.

Setting Map Ranges

Y To set the file and filter options

1. Click a map view to make it active.

2. From the Workspace Options toolbar, click Map View and choose Ranges from the
dropdown menu or right-click and choose Ranges.
By default, the map view is linked to the chromatogram.
3. To change the raw file and filter options, in the Map Ranges dialog box, select the release
check box.
4. From the File Name list, click the dropdown arrow to select a file name or click the
browse icon to select a different raw data file.
5. In the Filter box, type or select the scan filter from the dropdown list, which displays filter
options stored in the raw data file.
6. In the Retention Time and Mass boxes, enter a valid retention time and mass range
values.
7. To save the settings and close the Map Ranges dialog box, click OK.

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Saving a Map View

Defining the Retention Time and Mass Ranges

You can define the time and mass ranges in the Retention Time and Mass boxes as follows:
• Enter valid start and end numeric values for the RT and mass ranges.
–or–
• Set the start value as ‘*’ and enter a valid end value.
The application uses the default start value from the raw file as the start value.
–or–
• Defines a valid start value and enter the end value as ‘*’.
The application takes the default end value from the raw file as the end value.

Saving a Map View

You can save customized map views in the layout in the Freestyle application.

Y To save a customized Map View in the layout

1. Select the Map View that you want to save.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Layouts and choose an option to save the map view.
The Map View is saved in the layout.

When you apply the saved map view in another view, it displays details, such as title bar
number, state(s) (Dockable, Floating, Auto Hide), and the position of the map as it has been
saved in the layout. It also displays the map ranges parameters and the status of the Map View
(Linked or Released).

Setting the Map Display Options

Use the Display Options toolbar to set up the display options for a map view.

Y To open the Display Options dialog box for a map view

1. Click the Map View to make it active.

2. Click the Display Options tab to open the Display Options toolbar.
3. Follow one or more of these procedures to set up the map display options:
• Setting the Format Options
• Setting the Map Axis Options

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Setting the Map Display Options

• Setting the Map Color Options

• Setting the Band Width

Setting the Format Options

Use the options in the Format area of the Display Options toolbar to format the Map View.
Figure 111. Format area

Y To set the map format options

1. Click a map view to make it active.

2. Click the Display Options tab to open the Display Options toolbar.
3. To specify the arrangement style, select one of these options:
• To display a density map showing different shades for each intensity, click Plot
Options and choose Density from the dropdown menu.
The default display option is Density.
• To overlay plots vertically with optional horizontal skew (time offset) for the active
map, click Plot Options and choose Overlay from the dropdown menu.
The 3D view of the map appears.
4. To specify style options for overlaid (3D) plots, do the following in the Format area:
• To set the elevation angle (from 0 to 60 degrees), drag the Elevation slider or click the
left or right arrow on the Elevation slider until you reach the desired angle.
• To set the skew angle (from 0 to 45 degrees), drag the Skew slider or click the left or
right arrow on the Skew slider until you reach the desired angle.
The default values for the Skew and Elevation sliders are set at 30.
• To select a different filling style for the map view, select a fill option from the Fill list.
The default fill pattern is intensity shaded.
The styles control the display of filling and outlining data within the map. Outlining
(wireframing) produces lines between the scans (vertical or diagonal based on the
skew) and the mass bands (horizontal). Data with each scan and mass band is
displayed as either unfilled, filled with a solid color, or filled with a shade based on
the intensity of the data (using the shade colors as represented in the 2D density
map). Some modes draw a vertical line, which represents the intensity of each mass
band at each scan.

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 Table 39 explains the available styles, and how they control filling and outlining
(wireframe).

Tip In the table, nonzero data refers to data, which when scaled to a color, would
not provide a background color. When you want to view the smaller values of the
plot, press the UP arrow key on the computer to increase the intensity scaling or
set the log scale factor.

• To add a backdrop to 3D plots, click Draw Backdrop. To remove a backdrop, click

the Draw Backdrop icon again.
Table 39. Style options for overlaid (3D) plots
Modes Fill Frame Vertical Line
Plain Lines None None A vertical line at each scan and
mass/band having non-zero
data. All the lines are displayed
of same color. To differentiate
the lines, the bottom of each
line is highlighted in tinted
green.
Colored Lines None None A vertical line at each scan and
mass/band having non-zero
data. The line is colored based
on the intensity of the shade
selected from the color palette
None None Creates a wireframe plot, None
showing small amounts of
data that might otherwise
hidden by large data sets
Solid Color All non-zero bands are filled Creates a wireframe that None
using the same color helps to separate the bands
Intensity shaded All non-zero bands are filled None None
and colored based on the
shade selected from the color
palette.
Shaded with Frame All non-zero bands are filled Creates a wireframe that None
and colored based on the helps to separate the bands
shade selected from the color
palette.
 5 Reviewing Map Data
Setting the Map Display Options

Setting the Map Axis Options

The Map axis display options are based on the selected map view. For the Density view, the
default x-axis is Time (min) and the default for the z-axis is m/z. For the overlay 3D view,
there are three axes as follows:
• x axis – Time (min)
• y axis – Relative abundance
• z axis – m/z
Figure 112. Display Options – Map Axis
Show labels icons

Y To set the map axis options

1. Click a Map View to make it active.

2. Click the Display Options tab to open the Display Options toolbar.
3. To show or hide the labels of x, y, or z axis, click the corresponding Show Labels icons.
4. To display the grid lines, click Show Grid Lines.

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Setting the Map Display Options

Setting the Map Color Options

Use the color options in the Display Options toolbar to set the color of the traces.

Y To set the map color options

1. Click a Map View to make it active.

2. Click the Display Options tab to open the Display Options toolbar.
3. To select the color of the framing lines, from the Fill dropdown list in the Format area,
choose Plain Lines.
4. In the Color area, click the Line Color icon, .
The color palette lets you select a preset color or customize a color.
Figure 113. Color palette

5. Click the color you want to use for the line color, or click Advanced, create a custom
color, and click OK.
6. To select the color of the solid fill, from the Fill dropdown list in the Format area, choose
Solid Color.
7. In the Color area, click the Fill Solid icon, .
The color palette lets you select a preset color or customize a color. See Color palette.
8. To select the color of the backdrop (background), do the following:
a. In the Format area, click Plot Options and choose Overlay from the dropdown
menu.
b. In the Color area, click the Backdrop icon, .
The color palette lets you select a preset color or customize a color. See Color palette.
9. To select Gray Scale or color, do one of the following:
• To plot the map in gray scale, click the Gray Scale icon, .
• To plot the map in color, click the Gray Scale icon again.

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Setting the Map Display Options

10. Use the shade icons (0%, 20%, 40%, 60%, 80%, and 100%) to change the map’s color at
0%, 20%, 40%, 60%, 80%, and 100% relative abundance.
11. To select log scale or linear scale, do the following:
• To display the color of the map in a logarithmic scale, click the Log Scale icon. The
factor width that you set in the Factor box determines the scaling between color
bands.
• To display the map in a linear scale, click the Log Scale icon again.
12. To select smoothing, click Density view and then click Smoothing.
The solid color bands of the map view are converted to gradient color bands and the most
intense band appears at the middle and gradually fades out to the upward and downward
directions.

Setting the Band Width

Use the Band Width option to specify the bandwidth in amu units. The m/z axis of the map
view plot is directly related to the band width value entered in the m/z Band Width box. The
default band width is set at 1.0 amu.

The Map View does not display all the peaks in scans. It merges data into mass bands
displaying the most intense value in each scan. For example, when you scan a file from m/z
110 to 120, the map creates bands centered on each nominal mass, such as 109.5–110.5,
100.2–101.5, and so on. The view displays the most intense peak in each band. To increase
the mass resolution, reduce the band size.

Y To define the band width in a map view

1. Open a raw data file and make a map view the active view.
2. Click the Display Options tab to open the Display Options toolbar.
3. On the Band Width area, in the m/z Band Width (amu) box, type a value from 0.001 to
50.
Figure 114. Display Options – Band Width

4. To save the setting and close the dialog box, press the ENTER key on your keyboard.

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Setting the Map Display Options

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 6

Analyzing MS Data using Data Analytics View

Use the Data Analytics View display options in the FreeStyle application to analyze the MS
trending information from mass spectrometers. You can use the view to plot the chosen trace
type properties of an MS trending detector type. You can also analyze different aspects such as
frequency of occurrence, retention times, the quantity being plotted, categories, and so on.
The analysis is available as a Histogram or Trend report.

Contents
• Adding Data Analytics View to the Workspace
• Plotting the Data as a Histogram
• Data Analytics View - Specific Toolbars
• Saving a Data Analytics Plot
• Data Analytics Ranges

Adding Data Analytics View to the Workspace

You can add multiple data analytics views to the workspace.
Figure 115. Data Analytics View

Y To add a Data Analytics View

1. Open a raw data file (see Opening Raw Data Files or Sequence Files).
With the factory default layout, the Chromatogram view appears at the top and the
Spectrum view appears at the bottom.
2. In the Workspace Options toolbar, choose Data Analytics View – Insert View.

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6 Analyzing MS Data using Data Analytics View
Plotting the Data as a Histogram

A new Data Analytics view opens for the active chromatogram. A Histogram plot displays
the instrument status reading on the x axis and frequency on the y axis; and a Trend plot
displays the time on the x axis and readback parameter on the y axis.
When you have selected the MS Trending detector type, the Data Analytics View process
the information based on the selected trace type; otherwise, the first trace type is used to
process the information.
Note You can add multiple Data Analytics views in the workspace. When the Workspace
includes one or more Chromatogram views, a Data Analytics view of the selected or active
trace of the Chromatogram view appears.

Plotting the Data as a Histogram

When you create a new Data Analytics view with MS trending selected as the preferred trace
type, a Histogram plot displays the instrument status reading plotted against the frequency.
The data groups are represented as bins in the plot.
Figure 116. Histogram

The MS Trending detector type data is displayed as follows in the view:

• The vertical axis of the plot is Frequency (or Number of Occurrences). The title of
the vertical axis is Frequency.
• The horizontal axis of the plot is a binned representation of the data groups. The title
of the horizontal axis is the name of the readback parameter.
• When representing categorical data, the categories are plotted as bars on the
horizontal axis and number of occurrences are plotted on the vertical axis. In this
representation, bins are not created.
Note If the raw data file is a non-MS trending file, the application does not display a
histogram plot.

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Plotting the Data in a Trend Chart

Plotting the Data in a Trend Chart

When you plot the data in a Trend chart, the plot displays the trace type readback parameter
plotted against the time.

The MS Trending detector type data is displayed as follows in the view:

• The vertical axis of the plot is the readback parameter selected as the trace type. The
title of the vertical axis is the name of the selected readback parameter.
• The horizontal axis of the plot displays the readback trace during specific time period.

Data Analytics View - Specific Toolbars

Use the following Data Analytics view-specific toolbars to create, format, and set data
analytics ranges and display options of the view.
• Data Analytics View – Workspace Options Toolbar
• Data Analytics View – Display Options Toolbar

Data Analytics View – Workspace Options Toolbar

Use the Data Analytics view menu commands in the Workspace area of the Workspace
Options toolbar to create data analytics views and set the data analytics ranges.

Y To display the Data Analytics view-specific Workspace Options toolbar

1. Click the Chromatogram view to select it.

2. Click the Workspace Options tab to display the Workspace Options toolbar.
3. Click Data Analytics View and choose to create a new Data Analytics View or set the
data analytics ranges.
Figure 117. Data Analytics View-specific Workspace Options

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Data Analytics View - Specific Toolbars

Table 40. Data Analytics View – Workspace Options features

Command Description
Workspace area
Ranges Opens the Data Analytics Ranges dialog box to specify the
filename, detector type, trace type, and filter for the view.
Insert View Creates a data analytics view of the selected chromatogram.

Data Analytics View – Display Options Toolbar

Use the features in the Display Options toolbar to customize the Data Analytics view. You can
choose the plot type, define the number of bins and bar gap, and choose the color of the plot.

Y To display the Data Analytics View – Display Options toolbar

1. Click a Data Analytics view to select it.

2. Click the Display Options tab to open the Display Options toolbar.
The Data Analytics View-specific display options appear.
Figure 118. Data Analytics View – Display Options toolbar

Table 41. Data Analytics View – Display Options toolbar commands (Sheet 1 of 2)
Commands Description
Plot Type area
Histogram Displays a histogram plot view in the Data Analytics
View.
Trend Displays an MS trending plot view in the Data
Analytics View.
Data Filter area
All Values Displays all positive values and zeroes.
Positive Values and Zeroes Displays only positive values and zeroes.
Positive Values Only Displays only positive values.

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Saving a Data Analytics Plot

Table 41. Data Analytics View – Display Options toolbar commands (Sheet 2 of 2)
Commands Description
Format area
Number of bins Specifies the number of data groups to display.

Default: 50

Ranges: 1–1000

This does not apply to the Categorical data.

Bar Gap (%) Specifies the width of the gap between bars. The gap is
represented as a percentage of the width of the bar.

Default: 0–500
Log Scale Displays the bar in a logarithmic scale. When you click
the icon, logarithmic changes are applied to y-axis
values.
Color Displays the color palette where you specify the color of
the bars in the histogram plot.
Show Bar Ranges Displays the ranges of data represented by bars in the
histogram plot.
Decimals Specifies the required number of decimal places for the
bar range values.

Saving a Data Analytics Plot

You can modify and save the histogram or trend plots in the layout.

Y To save a customized Data Analytics View in the layout

1. Select the Data Analytics view that you want to save.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Layouts and choose an option to save the view.
The Data Analytics view is saved in the layout.

When you apply the saved Data Analytics plot in another view, the position of the bins is
displayed as it has been saved in the layout. When the imported layout has the binned
distribution of quantity plotted in a range that is not present in the current view, the
application displays the default distribution group.

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Data Analytics Ranges

Data Analytics Ranges

Use the Data Analytics Ranges option to create or update a Histogram or Trend plot.
Figure 119. Data Analytics Ranges dialog box

Table 42 describes the columns in the Data Analytics Ranges dialog box.
Table 42. Data Analytics Ranges dialog box
Column Description
File Name Specifies the location and file name of the raw data file with the
MS Trending data.

To import a new raw data file, use the browse icon to select a
different file.
Detector Type Specifies the detector used to acquire the data. By default, the
detector type is MS Trending.
Trace Type Specifies the trace type. For MS Trending, select one of the
instrument status parameters, such as the API source.
Filter Specifies the scan filter (same selections as the Scan Filters page
of the Info bar).

You can apply scan filters to filter the Trailer Extra information
and Custom trace scan time values. The Status trace type is not
associated with scans, and you cannot apply filters to the Status
trace type.

Filtering Data Analytics Views by Trace Types

You can create data analytics plots based on selected trace types. The different trace type
categories in the FreeStyle application are as follows:
• Status
• Trailer Extra
• Custom

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 6 Analyzing MS Data using Data Analytics View
Data Analytics Ranges

Figure 120. Data Analytics Plot Trace Types

Using the Status Trace Type

The first group in the Trace Type dropdown list is the Status trace type that displays the MS
detector status readback that was collected over time. You cannot apply filters to Status trace
types. It contains information about the different status of instrument, such as Temperature,
Pressure, Turbo Pump speed, and so on.

For example, Figure 121 shows the Trend plot created by the drugs_06 raw data file when the
MS Trending detector and Ambient Temperature trace type are selected. The plot displays
highest and lowest temperatures recorded between 28.4 oC and 28.55 oC due to the
temperature variations in the instrument.
Figure 121. Trends plot: Ambient Temperature

Figure 122. Histogram Plot: Ambient Temperature

Similarly, in Figure 123, the raw data file displays a histogram plot with one bar when Power
is selected. The x axis of the plot shows that the power remains static between 43.5 oC and
44.9 oC .

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Data Analytics Ranges

Figure 123. Histogram Plot: Power

Using Trailer Extra Information

The scan header displays important information about a scan, such as retention time, and
depends on the data acquisition settings for the mass spectrometer used to acquire the raw
data file. The detector can record additional information about each scan called Trailer Extra.
You can apply scan filters to filter the trailer extra information of each scan.

Figure 124 displays an MS2 scan filtered by number of ions.

Figure 124. Data Analytics Plot: Number of Ions

Using Custom Trace Type

The application includes the following Custom Trace types to filter an MS Trending scan:
• Scan Time
• MS Order
• Cycle Time

Scan Time: The amount of time required to accomplish one scan, from the lowest mass to the
highest mass of a specified scan range.

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 6 Analyzing MS Data using Data Analytics View
Data Analytics Ranges

Figure 125. Scan Time

When a Scan Time Trend plot is created, the scan header displays the total number of scans,
modal time, median time, average time, and average delta time for the selected data.
Table 43. Scan Time Trend Plot

Median time The median is a simple measure of central tendency. Median is

the middle value.
Average Time The average time taken per scan.
Average Delta Time Average of the time taken between two consecutive MS scans.

MS Order: The MS order of the mass spectra that makes up the chromatogram. It indicates
the order of the MS scan.
• MS1 is one MS scan.
• MS2 is an MS/MS scan.
• MS3 is an MS3 scan.

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6 Analyzing MS Data using Data Analytics View
Data Analytics Ranges

Figure 126. Data Analytics: MS Order Trace Type

Cycle Time: Cycle time is the time between two consecutive full MS scans. One cycle is the
start of one full MS scan to the start of the next/successive full MS scan.
Figure 127. Data Analytics: Cycle time trace type

Creating a Data Analytics Plot

Y To display a new plot for MS trending detector type

1. Click a Data Analytics View to make it active.

2. From the Workspace Options menu bar, choose Data Analytics – Ranges.
The Data Analytics Ranges dialog box opens with a list of the scans currently displayed in
the selected view.
3. In the Data Analytics Ranges dialog box, from the File Name list, click the dropdown
arrow to select a file name or click the browse icon to select a different raw data file.
In the Detector Type list, the MS Trending type is selected by default. You cannot edit
the detector type.
4. From the Trace Type dropdown list, select the trace type. For MS Trending, select one of
the instrument status parameters, such as the Ion Injection Time (ms).

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Data Analytics Ranges

5. In the Filter box, type or select a different scan filter from the dropdown list that displays
the filter options stored in the raw data file.
6. Click Apply, and then click OK.

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 7

Determining the Elemental Composition of Ions

To determine the elemental composition of ions, follow the procedures in these topics.

Contents
• Overview of an Elemental Composition Analysis
• Starting an Elemental Composition Analysis
• Using the Fragments Matching Algorithm for Confirmation
• Reviewing the Elemental Composition Results
• Modifying the Elements in the Elements in Use Table
• Using Custom Periodic Table for Analysis
• Elemental Composition Results View
• Simple Elemental Composition Results View
• Elemental Composition Page

Overview of an Elemental Composition Analysis

The FreeStyle application assigns chemical formulas to components by using an isotopic
pattern-matching algorithm that accounts for isotope accurate mass and intensity ratios.

The basic elemental composition algorithm uses a single mass, usually the monoisotopic mass
of a measured isotope pattern, to calculate all possible elemental compositions that lie within a
tolerance window. Then, the algorithm calculates a theoretical isotope pattern for each
elemental composition candidate. It calculates the fit between the theoretical and measured
isotope pattern, sorting the identified candidates in decreasing order of isotopic pattern score.
The isotopic pattern score value is a number between 0 percent (where the patterns are
completely different) and 100 percent (where the patterns are indistinguishable by using the
scoring parameters specified in the processing method).

The additional fragments-matching algorithm provides confirmation of the chemical formula

by comparing the fragmentation scan of the selected precursor mass to the theoretical
fragmentation pattern for the chemical formula.

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7 Determining the Elemental Composition of Ions
Overview of an Elemental Composition Analysis

To determine the elemental composition of a specific ion, you select the ion of interest in a
spectrum of interest, and then start the elemental composition analysis from the Workspace
Processing toolbar. To increase your confidence in the best matching formula, you can refine
the analysis by adding the information from a fragmentation scan for the selected ion.

When an elemental composition analysis finishes, the application displays the following:
• The Elemental Composition Results View, which lists the best matching chemical
formulas.
• The Elemental Composition Page of the Info Bar, where you can modify the parameters
that the elemental composition algorithm uses.
• A theoretical spectrum for the best matching formula in the Spectrum view or
MultiSpectrum view. The peaks in the theoretical spectrum are color-coded. Peaks above
the specified intensity threshold are green, and peaks below this threshold are red.

Figure 128 shows the Elemental Composition page and the Spectrum view with the
theoretical isotope pattern for the calculated elemental composition.
Figure 128. Elemental Composition page, Spectrum view, and Elemental Composition Results view

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 7 Determining the Elemental Composition of Ions
Starting an Elemental Composition Analysis

Starting an Elemental Composition Analysis

After you select a spectrum of interest, you can start an elemental composition analysis from
the Workspace Processing toolbar. When you start the analysis from the toolbar, you can
specify the analysis before the application processes the data.

Y To start an elemental composition analysis from the Workspace Processing toolbar

1. Click a Spectrum view to select it, or select the spectrum of interest in a MultiSpectrum
view.
For information about selecting a spectrum of interest, see Chapter 4, “Reviewing
Spectral Data.”
The Mass box at the top of the Elemental Composition page in the Info Bar is populated
with the m/z value for the selected spectrum’s base peak.
2. (Optional) To select another peak, double-click its m/z label, and then confirm the value
in the Mass box.
3. (Optional) To modify the settings for the advanced parameters, click Show Advanced
Parameters.
4. (Optional) To use a custom periodic table, select the Use Custom Periodic Table check
box, and then click Custom Table.
5. (Optional) Add the fragments matching algorithm to the analysis (see Using the
Fragments Matching Algorithm for Confirmation).
6. Click Calculate.
The application runs the elemental composition algorithm on the selected m/z value.

Using the Fragments Matching Algorithm for Confirmation

Use the Fragments Matching area of the Elemental Composition page to specify the
fragmentation scan number for the fragments matching algorithm. From the Workspace
Processing toolbar, you can specify the fragmentation scan before you start the analysis.

Y To use the fragments matching algorithm

1. Find an appropriate fragmentation scan by doing one of the following:

a. In the Info Bar, click the MSn Browser tab.
b. Expand the MS2 node for the precursor mass that you selected on the Elemental
Composition page.
c. Right-click the MSn Browser page, and choose Include Individual Scans.
Figure 129 shows the scan numbers of the individual scans for the MS2 precursor ion
at m/z 346.12.

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Figure 129. MSn Browser page showing the expanded list for m/z 346.11

–or–
a. In the Chromatogram view, insert an MS2 filtered chromatogram for the selected
ion.
b. Select the MS2 filtered chromatogram and use the left and right arrows to display a
data-dependent MS2 scan for the precursor ion in the Spectrum view.
2. On the Elemental Composition page, in the Mass box, check the m/z value of the selected
ion.
3. In the Fragments Matching area, select the Use Fragments Matching check box
(Figure 130).
Figure 130. Fragments Matching area of the Elemental Composition page

Advanced parameters

4. In the MSMS Scan No. box, type the scan number that you found in step 1.
5. Click Apply.

Review the results in the MSMS Coverage and MSMS Matched Peaks columns of the
Elemental Composition Results view.

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Reviewing the Elemental Composition Results

After you run an elemental composition analysis, you can review the results in the Elemental
Composition Results or Simple Elemental Composition Results views.

See these topics:

• Reviewing the Best-Matching Formulas
• Reviewing the MS/MS Coverage Score for Matching Fragments
• Reviewing MS/MS Annotation Results

Reviewing the Best-Matching Formulas

Review the results of an elemental composition analysis in the Elemental Composition Results
View. The application displays up to the maximum number of candidates specified on the
Elemental Composition page or the Default Elemental Composition page and orders the best
matching formulas by rank.

Y To review the best-matching formulas

1. For each row in the Elemental Composition Results view, review the formula, number of
matched isotopes, and MS coverage score.
2. Compare the theoretical isotope pattern for the proposed chemical formula, and then
zoom in on the mass peak of interest in the Spectrum view.
In the theoretical isotope pattern, a green marker represents the matched mass peaks that
fall inside the spectral distance and a red marker represents the missing mass peaks that
fall outside the spectral distance (mass tolerance plus intensity tolerance) at the
experimental resolution. An Orange marker represents the matched mass peaks that fall
outside the spectral distance in the Spectrum view.
The isotope pattern uses the resolution provided with the scan data to perform the
isotope pattern and calculate the spectral distance.

Figure 131 shows the elemental composition results for a mass peak at m/z 346.12.

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Figure 131. Theoretical isotope pattern with the markers

Theoretical isotope pattern
for the selected formula

You can view the chemical formula of each mass spectrum peak by clicking the Elemental
Comp. icon, , in the Spectrum – Display Options toolbar. You can also view the
theoretical mass and delta mass units of the simulated isotope profile when you point to the
isotope markers.
Note The data that displayed when you point to the isotope markers is different from the
Elemental Composition Results table. The results table displays the theoretical and delta
mass units for the monoisotopic peaks; pointing to the markers display data for the
simulated profile.

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Intensity Tolerances
The following figures define the tolerances and zones associated with elemental composition
results.
Figure 132. Identified tolerance bounds

Mass search
A spectrum peak is considered a match
when it is within these bounds (4x mass
tolerance). When no data is inside these
limits, this displays as red indicating that it
is not matched.
Intensity tolerance Mass tolerance
A spectrum peak might be a good A spectrum peak might be a good match
match when it is within these bounds. when it is within these bounds.

Isotope peak
Drawn at the theoretical isotope mass with
the width set by ± the mass tolerance.

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Figure 133. Zones

Green zone (not met)

A spectrum peak is considered a good
match when it is within a distance of 1.
Both the peak width (mass tolerance) and
Out of zone the intensity tolerance limits define a
This peak is out of zone for being a good distance of 1. An oval bounded by these
match to the peak to the right. It is also a tolerances traverses the distance=1 limit
partial match to the central peak. Because for a good match. (This annotation is not
such partial matches are hard to isolate, actually shown in the FreeStyle display.)
they do not improve scores.
See Green zone (met).

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Figure 134. Match data for the two orange peaks

Same peak matched (tentative).

Figure 135. Green zone (met)

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Modifying the Elements in the Elements in Use Table

Reviewing the MS/MS Coverage Score for Matching Fragments

After you run the elemental composition algorithm with the additional fragments matching
algorithm (see Using the Fragments Matching Algorithm for Confirmation), review the
results in the MS/MS Coverage [%] and MS/MS Matched Peaks columns of the Elemental
Composition Results table.

Y To compare the number of fragmentation peaks to the value in the MS/MS Matched
Peaks column
1. In a Spectrum view, display the fragmentation scan that you entered on the Elemental
Composition page, in the Fragments Matching area, in the MS/MS Scan No box.
2. Compare the values for MS/MS Coverage [%] and MS/MS Matched Peaks to the
number of mass spectrum peaks in the fragmentation scan.

Reviewing MS/MS Annotation Results

After you perform the elemental composition, you can review the results of an MS/MS scan in
the MSMS Annotation result window. The application displays the result when you type the
spectral scan corresponding to the MS/MS scan number in the info bar.

Y To review the MS/MS annotation results

1. The spectral scan corresponding to MS/MS scan number opens only when you enable the
Use fragment matching option and type a valid MS/MS scan number in the info bar.
2. When you change the MS/MS scan number and re-perform the elemental composition
analysis, the result of the MS/MS scan is automatically replaced in the MS/MS
Annotation result window.
3. After completing the Elemental composition analysis, the MS/MS Annotation result
window is annotated with the elemental composition of the fragment masses.
Note Zoom in on the MS/MS Annotation Result page to view the annotation of the
lower intensity peaks.

Modifying the Elements in the Elements in Use Table

The elemental composition algorithm uses only the minimum and maximum number of
atoms for each element in the Elements in Use table to determine the elemental composition
of ions. You can edit the Elements in Use table in two locations: the Elemental Composition
page (in the Info Bar) or the Default Elemental Composition page (in the Default Options
Configuration dialog box).
To add elements to or remove elements from the table, follow the appropriate topic:
• Adding Elements to the Elements in Use Table
• Elemental Composition Results View

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Modifying the Elements in the Elements in Use Table

Adding Elements to the Elements in Use Table

Use the Periodic Table of Elements dialog box to add isotopes to the Elements in Use table on
the Elemental Composition page or Default Elemental Composition page.

Y To add an element to the Elements In Use table

1. To display the dialog box of the periodic table of elements, on the Elemental
Composition page or the Default Elemental Composition page, click Add below the
Elements in Use table.
Figure 136. Periodic table of elements

2. Do the following for each element that you add:

a. Click the element’s symbol in the table.
The element’s isotopes appear below the periodic table.
b. When the selected element has several isotopes, select the isotope with the highest
abundance for non-labeled analytes or the appropriate isotope for labeled analytes.
c. Specify the limits for the element as follows:
• In the Min box, type an integer to specify the minimum number of atoms of this
element that must be in the calculated elemental composition.
• In the Max box, type an integer to specify the maximum number of atoms of this
element that can be in the calculated elemental composition.
d. Click Add to List.
3. Click OK to add the selected elements to the table.

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Removing Elements from the Elements In Use Table

Y To remove an element from the Elements In Use Table

1. On the Elemental Composition page in the Info Bar or the Default Elemental
Composition page in the Default Configuration Options dialog box, below the Elements
in Use table, click to the left of the element to highlight its row (Figure 137).
Figure 137. Elements in Use area on the Elemental composition page of the Info Bar

Selected row

2. Click Remove, and in the confirmation box, click OK.

Using Custom Periodic Table for Analysis

Use the Custom Periodic table for the elemental composition calculation by adding custom
isotope abundance to the elements. The table provides a list of all known isotopes, including
those having zero abundance. When you select an element from the list, the elemental
composition details of the selected element display separately. You can edit the abundances of
each isotope and change the total abundance of the element. The abundance of each isotope
must be greater than 0 and the total abundance must be equal to 1. You can save the changes
and also load an existing custom periodic table.
Figure 138. Custom Periodic Table

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Using Custom Periodic Table for Analysis

Y To add custom isotope abundance

1. On the Elemental Composition page in the Info Bar, below Custom Periodic Table, select
the Use Custom Periodic Table check box.
2. Click Custom Table.
3. Click the element’s symbol in the table.
The element’s isotopes appear below the periodic table.
4. Select the isotope and edit the abundance.
5. Click Save.
The Elemental Composition page displays the name and path of the loaded custom
periodic table.

Note The application highlights the Total Abundance value column in a red rectangle
if the abundance is greater or less than 1.

6. Click Revert to Default to revert and use the default periodic table.
7. Click Load to use an existing custom periodic table saved in your computer.

In the following example, when N is selected from the Element list, the isotopes 14 and 15 are
displayed below with an abundance of 0.99632 and 0.00368 respectively. The Total
Abundance of the isotopes is 1.
Figure 139. Total Abundance equals to 1

When the abundance of isotope 14 is changed to 0.89632, the Total Abundance becomes 0.9
and the value is highlighted in a red rectangle as the abundance is less than 1.

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Elemental Composition Results View

Figure 140. Total Abundance less than 1

When you run an elemental composition analysis using the custom periodic table, the
application displays the matching chemical formula for the selected peak in the spectrum
based on the modified abundance. The application uses default periodic table if the custom
periodic table is not selected for analysis.

Elemental Composition Results View

After you run an elemental composition analysis using the Pick Peak parameter, the Elemental
Composition Results view appears with the best matching calculated chemical formulas for
the selected peak in the spectrum.

Table 44 describes the columns in the Elemental Composition Results view. By default, the
S Fit, RDB, Combined Score, MSMS Cov. [%], MSMS Shift Measure, and MSMS Matched
Peaks columns are hidden. To display these columns, use the Field Chooser dialog box.
Table 44. Elemental Composition Results view parameters (Sheet 1 of 3)
Column Description
Field Chooser icon ( ) Opens the Field Chooser dialog box for selecting the result
columns to display.
Peak Mass Displays the mass-to-charge ratio of the selected isotopic peak.
Display Formula Displays the chemical formula of the match by using the elements
that you specified on the Elemental Composition Page. Also
displays isotope labels for elements listed in the Elements in Use
table.

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Table 44. Elemental Composition Results view parameters (Sheet 2 of 3)

Column Description
S Fit Displays the spectral similarity score between the measured and
theoretical isotope patterns. Fragments matching does not affect
this value.
RDB Displays the ring and double-bond (RDB) equivalents that the
algorithm calculated for the proposed match.
Delta (ppm) Displays the difference between the measured mass-to-charge ratio
and the theoretical mass-to-charge ratio. The table lists only
formulas whose mass is within the tolerance that you specified on
the Elemental Composition Page, in the units (ppm, mmu, or
amu) that you specified there.
Theoretical Mass Displays the theoretical mass-to-charge ratio of the proposed
match.
Rank Displays the ranking of the proposed match by decreasing
Combined Score values.
Combined Score Displays a percentage that conveys how close the measured
spectrum matches the theoretical spectrum. Fragments matching
refines this value by including the relative number of matching
fragments in the score. The Combined Score for a proposed
formula increases if the fragmentation spectrum includes more
matching fragments and decreases if the fragmentation spectrum
includes fewer matching fragments relative to other formulas.
#Matched Isotopes Displays the number of isotopes in the measured isotope pattern
that match the theoretical isotope pattern for the chemical
formula.

A matching isotope matches the delta mass from the A0 peak (the
peak with the lowest m/z value in an isotope pattern) and the
relative intensity of the theoretical isotope pattern within the
specified tolerances.
#Missed Isotopes Displays the number of isotopes that are missing in the measured
isotope pattern for the precursor ion. An isotope peak is missing if
it is part of the theoretical isotope pattern and not found in the
measured spectrum within the specified mass and intensity
tolerances.

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Table 44. Elemental Composition Results view parameters (Sheet 3 of 3)

Column Description
MS Coverage [%] Displays the summed intensity of matching isotope peaks in the
measured pattern relative to the summed intensity of all the peaks
in the measured pattern.
Summed intensity of the matching isotope peaks × 100
--------------------------------------------------------------------------------------------------------------------------------
Summed intensity of all the peaks in the measured pattern
IMPORTANT Low values for all the candidates might indicate
an overlapping pattern rather than a lack of good matches.
Pattern Coverage [%] Displays the summed intensity of the matching isotope peaks in
the measured MS1 spectrum relative to the summed intensity of
the theoretical isotope pattern.
Summed intensity of the matching isotope peaks × 100
--------------------------------------------------------------------------------------------------------------------------
Summed intensity of the theoretical isotope pattern

Provides a quantitative measure of how well the measured isotope

pattern matches the theoretical isotope pattern.
Note Because the A0 peak (peak with the lowest m/z value in
the pattern) is typically responsible for most of the pattern
intensity, even a small decrease in the percent coverage might be
important. For example, a missing peak for an isotope with two
13C atoms might cause only a small decrease in the summed

intensity of the measured isotope pattern.

The following columns display nonzero values when you add the fragments matching
algorithm to the elemental composition analysis.
MSMS Cov. [%] Displays the summed intensity of the matched fragment peaks
relative to the summed intensity of all the fragment peaks in the
selected MSMS scan.
Note Low values for all the candidates might indicate a
contaminating compound within the isolation window for the
fragmentation scan.
MSMS Shift Measure Not used for scoring.

The application searches for each fragment in a range from the

expected mass based on the chemical formula and the expected
mass plus the delta mass between the precursor ion and its
displayed formula. The MSMS Shift Measure value increases
when the fragments are closer to their expected masses rather than
their shifted masses.
MSMS Matched Peaks Displays the number of matching fragments in the MSMS
spectrum. Click the expand icon, +, to view the fragment list.

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Simple Elemental Composition Results View

Simple Elemental Composition Results View

After you run an elemental composition analysis using the Mass parameter, the Simple
Elemental Composition Results view appears with the best matching calculated chemical
formulas in the spectrum.

Table 45 describes the columns in the Simple Elemental Composition Results view.
Table 45. Simple Elemental Composition Results view parameters
Column Description
Searched Mass Displays the mass identified in the Mass box on the Elemental
Composition page.
Formula Displays the chemical formula of the match by using the elements
that you specified on the Elemental Composition Page.
Mass Displays the mass-to-charge ratio of the match.
RDB Displays the ring and double-bond (RDB) equivalents that the
algorithm calculated for the proposed match.
Delta (ppm) Displays the difference between the measured mass-to-charge ratio
and the theoretical mass-to-charge ratio. The table lists only
formulas whose mass is within the tolerance that you specified on
the Elemental Composition Page, in the units (ppm, mmu, or
amu) that you specified there.

Y To export simple elemental composition results as a CSV file

1. Click the Simple Elemental Composition Results view to select it.

2. From the Workspace Options toolbar, choose Exports > Export Selection As.
3. In the Copy to Clipboard/Export dialog box, select the To CSV Files option and click
OK.
4. In the Export Data dialog box, type a name for the CSV file and click Save.
The Excel application opens and displays the results data in the following format.

Note To export the entire workspace including the simple elemental composition
results, choose Exports > Export Workspace As.

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Elemental Composition Page

Use the Elemental Composition page (Figure 141) in the Info Bar for specifying the criteria to
calculate the best matching chemical formula for a mass-to-charge ratio in a mass spectrum.

To display advanced parameters, click Show Advanced Parameters.

Figure 141. Elemental Composition page

Without advanced parameters

With advanced parameters

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Elemental Composition Page

Table 46. Elemental Composition page parameters (Sheet 1 of 5)

Parameter Description
Show/Hide Advanced The advanced parameters to show or hide are as follows:
Parameters
• Under Prediction Settings: Min and Max RDBE, Nitrogen Rule, Centroid
Algorithm, Normalization Mode, and Use Representative Elements
• Pattern Matching: Intensity Threshold and Intensity Tolerance
• Under Fragments Matching: S/N Threshold and Min. Spectral Fit
Mass
Mass Specify mass (in the Mass box) for elemental composition analysis.

Range: 0.5 to 100 000

Pick Peak Select the mass peak from the spectrum for elemental composition, isotope pattern
matching, and fragments matching analysis.
Pick Scan Applies elemental composition analysis for all mass peaks in the spectrum. The
spectrum plot and each row of the spectrum list displays the highest scoring result for
each peak. The results view is not available for this analysis.
Calculate Calculate formulas and display them in the Elemental Composition Results View.
Prediction Settings
Mass Tolerance Specify a mass tolerance to restrict the number of possible elemental compositions.

The elemental composition algorithm returns results of the search only if the
theoretical mass matches the submitted mass within the specified tolerance.

Range: 0.00 to 100.00; default: 5.00 ppm

Units Select the units that you want to associate with the mass tolerance.

The options are amu (atomic mass units), mmu (millimass units), and ppm
(parts-per-million). When you specify the error limits in ppm, the errors are
mass-dependent and get larger at higher masses and smaller at lower masses.

Default: ppm
Max #Candidates Specify the maximum number of formulas to display.

Range: 0 to 400; default: 10

Charge Select the charge state that you want to use to calculate the probable formulas.

Range: –99 to 99; default: 1

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Table 46. Elemental Composition page parameters (Sheet 2 of 5)

Parameter Description
Min. and Max. RDBE Specify a range of values for double bonds and ring equivalents—a measure of the
number of unsaturated bonds in a compound—that limits the calculated formulas to
only those that make sense chemically.

Limits range: –1000.0 to 1000.0

The value is calculated by the following formula:

imax

 Ni ( Vi – 2 )
i
D = 1 + ------------------------------------------
2

where:
• D is the value for the RDB equivalents
• imax is the total number of different elements in the composition
• Ni is the number of atoms of element i
• Vi is the valence of atom i

The calculation produces an integer such as 3, which indicates an odd-electron ion, or

a number with a remainder of 0.5, which indicates an even-electron ion.

Minimum value: –0.5, corresponding to a protonated, saturated compound

Nitrogen Rule Select whether or how to use the Nitrogen Rule in the formula calculation:
• Do Not Use: Do not use the Nitrogen Rule.
• (Default) Even Electron Ions: Select for even-electron ions, such as protonated
species.
• Odd Electron Ions: Select for odd-electron ions, such as radical cations.
Note McLafferty states the Nitrogen Rule as follows: “If an odd-electron ion
contains no (or an even number of ) nitrogen atoms, its molecular ion will be at an
even mass number... [Similarly,] an odd-electron ion will be at an odd mass number
if it contains an odd number of nitrogen atoms.”
Centroid Algorithm Select the centroiding algorithm for profile data.

Default: FT Orbitrap
Selections: FTOrbitrap, GCQ, TSQ, or MAT

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Table 46. Elemental Composition page parameters (Sheet 3 of 5)

Parameter Description
Normalization Mode Select one of the following normalization modes for the spectral peak intensities:
• (Default) Base peak: The most common normalization mode. Normalizes the
isotope peaks to the base peak height of 100%. Its disadvantage is the propagation
of the intensity error of the base peak to all isotope intensities.
• Linear: Normalizes the theoretical and measured patterns so that the sum of all
isotopic pattern intensities is the same.
• Quadratic: Normalizes the theoretical and measured patterns so that the error
squares (intensity differences) are minimized.
Use Representative Elements Select the check box to use the Representative Elements table for the elemental
composition calculations. Clear the check box to use the Protein Elements table.

Currently the elemental composition algorithm supports two types of element tables,
differing mainly in their carbon (C) abundances.
• The Representative Elements table has the following C abundances:
– 12C: 0.9893
– 13C: 0.0107
• The Protein Elements table has the following C abundances:
– 12C: 0.989136445
– 13C: 0.010863555

Default: Selected
Pattern Matching
Intensity Threshold [%] Specify the isotope intensity threshold, relative to the base peak of the theoretical
isotope pattern that the algorithm uses for pattern simulation. The algorithm skips
isotopes below the threshold—that is, if the expected intensity of an isotopic peak is
below the threshold, the algorithm does not look for the peak in the experimental
spectrum, and the peak is not part of the score calculation.

Range: 0.00 to 10 000.00%; default: 0.10%

Intensity Tolerance [%] Specify the relative intensity tolerance for the isotope search.

Range: 0.00 to 10 000.00%; default: 30.00%

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Table 46. Elemental Composition page parameters (Sheet 4 of 5)

Parameter Description
Fragments Matching
Use Fragments Matching Select this check box to turn on the fragment matching algorithm, which ranks the
identified candidates (chemical formulas) by the number of matching peaks in the
fragmentation scan for the precursor ion.

The fragment matching algorithm requires a fragmentation scan for the selected
precursor ion.

To perform MS/MS matching, you must enter the scan number of the fragmentation
scan for the precursor ion (m/z value) of interest.
Mass Tolerance Specify the mass tolerance for the peaks in the fragmentation spectrum (expected mass
versus theoretical mass).
MSMS Scan No Specify the MS/MS scan number of the fragmentation scan for the selected precursor
ion.

For instructions on how to find an appropriate scan number, see Using the Fragments
Matching Algorithm for Confirmation.

Default: 0 (No fragment matching); range: 1 to the last scan number in the raw data
file
S/N Threshold Specify the signal-to-noise threshold for the peaks in the MS/MS spectrum. The
fragments matching algorithm ignores peaks below this S/N threshold.
Min. Spectral Fit [%] Restrict the candidate list to candidates that meet or exceed the minimum spectral fit
value.

Range: 0.00 to 100.00%; default: 10.00%

Custom Periodic Table

Defines elements having customized isotope abundance that you want to consider when the algorithm calculates the
elemental composition.
Use Custom Periodic Table Use the custom periodic table for elemental composition analysis.
Button
Save Saves the modified isotope abundances.
Save As Saves the custom periodic table to your computer.
Load Selects an existing custom periodic file that contains the modified isotope abundances
to consider for the elemental composition analysis.

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Table 46. Elemental Composition page parameters (Sheet 5 of 5)

Parameter Description
Elements in Use

Lists the isotopes and the number of occurrences for each isotope to consider when the algorithm calculates possible
elemental compositions for the submitted mass value.

See Modifying the Elements in the Elements in Use Table.

Field Chooser icon ( ) Displays the Field Chooser dialog box for selecting which fields appear in the Elements
in Use table (see Selecting the Columns to Display in a View or Dialog Box with
Tabular Data).
Isotope Select the isotopes that you want the data system to consider when it calculates the
possible elemental compositions for a monoisotopic ion with the given mass (A0 mass
peak).

To add an isotope, click Add. The dialog box for the periodic table of elements opens.
You can also right-click in the grid and choose Add from the shortcut menu.

To remove an isotope, click to the left of the element in the table to highlight its row,
and then click Remove or right-click and choose Remove from the shortcut menu.
Min Specify the minimum number of occurrences of an isotope in the chemical formula.
Max Specify the maximum number of occurrences of an isotope in the chemical formula.
Mass Displays the exact isotopic mass for each isotope in the Elements in Use list.

You cannot edit this value.

Simulated Spectrum
Profile Select the Profile check box to add an overlay of the simulated spectrum. The
Resolution box displays the value used to perform the elemental composition. You
cannot edit the resolution value. The scores are calculated based on the peaks
determined from the profile.
Tip You can view the resolution values for each mass spectrum peak by clicking Peak
Resolution in the Spectrum – Display Options toolbar (see Spectrum – Display
Options Toolbar).
Button
Load Select a file (with an .limx file name extension) that contains a set of isotope limits.
Save As Save a list of isotope limits to a file (with an LIMX file name extension).
Apply Apply the elemental composition settings to the active mass spectrum.
Help Opens the FreeStyle Help to the elemental composition topic.

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 8

Searching Mass Spectrum Libraries

To compare a query spectrum against a set of mass spectrum libraries, follow the procedures in
these topics.

Contents
• Performing a Local NIST or mzVault Library Search
• Reviewing the Results of a Local NIST or mzVault Library Search
• Modifying the Settings for a Local NIST or mzVault Library Search
• Searching the Online mzCloud Mass Spectral Database
• Exporting a Mass Spectrum to the NIST MS Search Application
• Managing Libraries
• Spectrum Workspace Processing Toolbar – Library Search Features

Performing a Local NIST or mzVault Library Search

Search your local NIST or mzVault mass spectrum libraries to identify unknown compounds.

Follow the procedures in these topics in order:

1. Setting Up the Default Library Search Parameters
2. Selecting the Query Spectrum
3. Starting a Library Search

Setting Up the Default Library Search Parameters

Before you can perform an mzVault library search, you must specify the location of at least
one of your local mzVault database files. The application automatically links to the NIST
libraries installed during the software installation process.

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8 Searching Mass Spectrum Libraries
Performing a Local NIST or mzVault Library Search

Follow these procedures:

• To open the Library Search page of the Default Options Configuration dialog box
• To specify the location of your local mzVault database files
• To specify the default parameter settings for a library search

Y To open the Library Search page of the Default Options Configuration dialog box

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Library Search.

Y To specify the location of your local mzVault database files

1. In the Library Type area on the Library Search page, select the mzVault option.
2. For each database file that you want to add to the Search list, do the following:
a. In the Search List area, browse to the location of your mzVault database files.
b. Select an mzVault database file (DB).
c. Click Open.
3. Click Save.

Tip After you run your first mzVault search, you can also specify the location of your
mzVault database files on the mzVault Search page in the Info Bar.

Y To specify the default parameter settings for a library search

1. In the Library Type area on the Default Library Search page, select either the NIST or
mzVault option.
2. Make the appropriate selections and entries.
3. Click Save.

Note If you plan to run both NIST and mzVault searches, make sure to select and
save the settings for both search types.

Tip After you run a library search, selecting a different setting from the Display list on
the mzVault Search page or the NIST Search page (in the Info Bar) automatically
changes how the Spectrum view displays the query spectrum versus the matching
library spectrum. You can also modify the search criteria from the Info Bar; however,
to start the new search, you must click Apply.

Go to the next topic “Selecting the Query Spectrum.”

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Selecting the Query Spectrum

The query spectrum is the spectrum that you want to search against a library of mass spectra.

Y To select the query spectrum

1. Open a raw data file that contains mass spectral data.

IMPORTANT You can perform a library search on a mass spectrum, an average mass
spectrum, or a composite mass spectrum. For an mzVault or a NIST search for a
matching MS/MS spectrum, the query spectrum must be a data-dependent scan.

2. Select a mass spectrum by doing any of the following:

• Select a data point in a chromatogram plot. If a Spectrum or MultiSpectrum view is
not open, in the Workspace Options toolbar, click Spectrum.
The mass spectrum for the selected time point appears in the Spectrum view or the
MultiSpectrum view.
• Click the MSn Browser tab, and then double-click an item in the MSn Tree.
The selected spectrum opens in a new Spectrum view.
–or–
a. Click the Spectrum (or MultiSpectrum) to select it.
b. In the Workspace Options toolbar, click Spectrum and choose Ranges from the
dropdown menu.
c. In the Spectrum Ranges dialog box, specify the spectrum of interest.

Note You can perform a library search on a mass spectrum, an averaged mass
spectrum, or a composite mass spectrum.

Go to the next topic “Starting a Library Search.”

Starting a Library Search

After you specify the default options for an mzVault or NIST library search and select a query
spectrum, you are ready to start the search.

Y To start a library search

1. Specify the default search settings (see Setting Up the Default Library Search Parameters).
2. Select a spectrum plot (see Selecting the Query Spectrum).
3. Click the Workspace Processing tab to open the Workspace Processing toolbar.

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4. Click Library Search and choose mzVault Search or NIST Search from the dropdown
menu.
The selected library search page appears in the Info Bar, and the application automatically
searches the specified libraries.
When the search ends, the following items appear:
• The library spectrum for the best hit appears in the Spectrum view as a stacked plot, a
mirror plot, or an exclusion plot.

Note From the Info Bar, you can change the display setting on the mzVault
Search page or the NIST Search page without rerunning the search.

• A list of search hits appears in the search results view.

• The structure of the best hit appears in the chemical structure view.

Go to the next procedure “Reviewing the Results of a Local NIST or mzVault Library Search.”

Reviewing the Results of a Local NIST or mzVault Library Search

To review the results of a library search, see the appropriate topics:
• Reviewing the Results of a NIST Library Search
• NIST Search Results View
• Reviewing the Results of an mzVault Search
• mzVault Search Results View

Reviewing the Results of a NIST Library Search

Figure 142 shows the results of a NIST library search. The Spectrum view contains a mirror
plot. The NIST Search Results view lists 10 hits sorted in order of probability. The Chemical
Structure View displays the two-dimensional structure of the selected hit.

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Figure 142. Results of a NIST library search displayed as a mirror plot

Query
spectrum

Library
spectrum

NIST Search Results View

The NIST Search Result view appears in the workspace after you submit a NIST library
search. The library search algorithm returns a list of the best matches from the selected
libraries. See Managing Libraries.

Y To display the NIST Search Results view

Perform a NIST library search (see Performing a Local NIST or mzVault Library Search).

Table 47 describes the columns for the NIST Search Results view.
Table 47. NIST Search Result view columns (Sheet 1 of 2)
Column Description
Hit Relative ranking of library search matches based on decreasing SI
(Search Index) values.
SI (Search Index) Direct matching factor for the query spectrum and the library
spectrum.
RSI (Reverse Search Reverse search matching factor that ignores any spectrum peaks in
Index) the query spectrum that are not in the library spectrum.
Prob Probability factor based on the differences between adjacent hits
in an SI ordered list.

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Table 47. NIST Search Result view columns (Sheet 2 of 2)

Column Description
Molecular Weight Molecular weight (in daltons) of the library search match.
Chemical Formula Chemical formula of the library search match.
Name Name of the matched compound in the library.
Library Name Name of the library that contains the matching compound.
NIST Record Number Record number of the matched compound in the NIST library.
Score Number of results returned from NIST.
Dot. Prod. Defines the score returned from the NIST library.
Delta Mass Delta Mass of the matched compound in the library. It is the
differences between the experimentally determined mass-to-charge
ratios and the theoretical mass-to-charge ratios. Displays delta
mass only for the MS/MS Hybrid search type.

Three factors describe the accuracy of the match to the submitted spectrum: SI, RSI, and
Prob. With the SI and RSI matching factors, a perfect match results in a value of 1000. As a
general guide, 900 or greater is an excellent match; 800–900, a good match; and 700–800, a
fair match. A matching factor less than 600 is a poor match. Unknown spectra with many
peaks tend to yield lower match factors than similar spectra with fewer peaks.

The probability factor is a complex parameter based on the SI matching factor and the
difference between adjacent matches. If a match has an SI match factor greater than 900 and
the next best match has a match factor of 300, the probability of the compound being
correctly identified is high. Conversely, if several matches have very similar SI matching
factors, the probability of a correct assignment is low.

The Chemical Structure View appears when you perform a NIST library search. The
Chemical Structure view displays the chemical structure, the compound name, and the
chemical formula of the compound that you select in the NIST Search Result view.

Reviewing the Results of an mzVault Search

For information about starting an mzVault library search, see Starting a Library Search.

Follow these procedures:

• To review the matching compounds found by an mzVault library search
• To view all the matching scans for a selected compound

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Y To review the matching compounds found by an mzVault library search

In the mzVault Search Results view, select a compound.

The best matching spectrum for the library compound appears in the Spectrum view and
its chemical structure appears in the mzVault Chemical Structure View.
Figure 143 shows the results of an mzVault library search. The query spectrum’s plot is
stacked above the library spectrum’s plot. The mzVault Search Results view lists matching
compounds in order of their compound ID numbers. The mzVault Chemical Structure
view displays the two-dimensional structure of the compound with the highest score,
from 0 to 100%.
Figure 143. mzVault library search results

Query spectrum

Library spectrum

mzVault Search
Results view
sorted in
ascending order
by Compound ID

Structure of
selected
compound

Expand icon

Y To view all the matching scans for a selected compound

1. Click the expand icon, , to open the complete list of matching scans.
2. To view a scan in the Spectrum view, select it in the subtable.

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Figure 144 shows the subtable of matching scans in the mzVault database file. For details
about the table columns, see the next topic “mzVault Search Results View.” To modify
and rerun the search, see To reset the mzVault search parameters and query spectrum.
Figure 144. Subtable of matching scans in the mzVault Search Results view

mzVault Search Results View

The mzVault Search Results view appears in the workspace after you submit an mzVault
search. The mzVault search returns a list of matching compounds.

Y To display the mzVault Search Results view

Perform an mzVault library search (see Performing a Local NIST or mzVault Library
Search).

The mzVault Search Results view consists of the top-level compound entries and the
secondary-level spectrum entries for each compound hit.

Table 48 describes the columns in the compounds table in the mzVault Search Results view.
Table 48. Compounds table in the mzVault Search Results view (Sheet 1 of 2)
Column Description
Compound ID Displays the assigned entry number in the mzVault library. The
mzVault application assigns entry numbers in sequential order,
beginning with the number 1 for the first entry.
Compound Displays the name of the compound associated with the matching
spectrum in the library.
Formula Displays the chemical formula for the library entry.

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Table 48. Compounds table in the mzVault Search Results view (Sheet 2 of 2)
Column Description
CAS ID Displays the unique Chemical Abstracts Service™ registry number.
High Res Score Displays a score calculated by a proprietary algorithm that
indicates how well the library spectrum and the query spectrum
match.

Table 49 describes the spectrum information for each compound entry.

Table 49. Spectrum information for each compound entry
Column Description
Spectrum ID Displays the identification number for the library spectrum.
Filter Displays the scan filter from the raw data file.
RT Displays the retention time from the raw data file.
Scan Number Displays the scan number from the raw data file.
Precursor m/z Displays the mass-to-charge ratio of the precursor ion from the
scan filter for the library spectrum.
Neutral Mass Displays the uncharged, neutral mass of the molecule.
Delta Mass Displays the difference in mass between the mass-to-charge ratio
of the precursor ion for the library entry and the mass-to-charge
ratio of the precursor ion for the query spectrum, in parts per
million.
High Res Score Displays the score calculated by a proprietary algorithm that
indicates how well the library spectrum and the query spectrum
match.
Raw File URL Displays the name and path of the raw data file where the
matching spectrum in the library entry came from.

Modifying the Settings for a Local NIST or mzVault Library Search

After you run a NIST or an mzVault library search, you can modify the search settings on the
respective page in the Info Bar, and then restart the search.

To modify and rerun a search, see the appropriate topic:

• Modifying a NIST Search from the NIST Search Page
• Modifying an mzVault Search from the mzVault Search Page

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Modifying a NIST Search from the NIST Search Page

After running a NIST search, use the NIST Search page of the Info Bar to modify the search
or the display in the NIST Search Results view.

Follow these procedures:

• To display the NIST Search page
• To modify the display in the NIST Search Results view
• To modify the NIST search

Y To display the NIST Search page

Run a NIST library search (see Performing a Local NIST or mzVault Library Search).
When the search is complete, you can reset the search parameters.
Figure 145 shows the NIST Search page.
Figure 145. NIST Search page

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Y To modify the display in the NIST Search Results view

In the Display list on the NIST Search page, make a different selection (see Display in
Table 50).

Y To modify the NIST search

Modify the parameter settings on the NIST Search page, and then click Apply.

Table 50 describes the parameters for the NIST Search page.

Table 50. NIST Search page parameters (Sheet 1 of 4)
Parameter Description
Search List

Use the settings in this area to choose the library for library searches. Specify how many search results to display and
how they are displayed.
Library Select the search libraries.
Hits Select how many search results to display in the NIST Search Results View.

Range: 1–100; default: 10

Display Select how to display the library spectrum and measured spectrum, from these options:
• Stacked: Displays the submitted spectrum above the currently selected library spectrum in
the match list.
• Exclusion: Subtracts the library spectrum from the submitted spectrum.
• Mirror: Plots the submitted spectrum that points up from the x axis and the library
spectrum that points down from the x axis.
Search Type

Use the options in this area to choose the type of library search to apply. The two main search types are Identity and
Similarity. They differ primarily in the weightings of the spectrum as a function of mass.
Identity Applies an identity-search algorithm for the library matching of spectra.
Normal (Default) Applies a normal identity-search algorithm for library matching of spectra.

Use a normal identity search for low quality or unusual spectra. The search algorithm uses a
standard prescreen search filter.
Quick Applies a quick identity-search algorithm for library matching of spectra.

Use this option if you are sure the spectrum or compound exists in the library. The search
algorithm uses a fast prescreen search filter.
MS/MS Searches for an MS/MS spectrum in a library of MS/MS spectra.

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Table 50. NIST Search page parameters (Sheet 2 of 4)

Parameter Description
In-source HiRes Searches for an in-source electron ionization (EI) spectrum in a library containing
high-resolution in-source EI spectra—that is, a library where the EI-MS spectra are annotated
with m/z values to several decimal places. Also searches for an MS/MS spectrum in a library of
MS/MS spectra.

Unlike an MS/MS search, this search does not compare precursor m/z values.
Similarity Applies a similarity-search algorithm for the library matching of spectra.
Simple Applies a simple similarity-search algorithm for library matching of spectra.

This option finds a large set of spectra to compare with the submitted spectrum, and is
generally slower than an identity search.

Use a simple similarity search in these situations:

• You know that the unknown spectrum is not in the library.
• The spectrum is of poor quality so that a reliable match is unlikely.
Hybrid Applies a hybrid similarity-search algorithm for library matching of spectra.

This option uses a combination of the simple and neutral-loss search strategies. The
neutral-loss search requires an estimate of the unknown’s molecular weight. When the
unknown compound contains chemical structures that generate both characteristic ions and
neutral loss patterns, the search algorithm can identify these structures from the match list
produced by this search.
Neutral Loss Applies a neutral-loss similarity-search algorithm for library matching of spectra.

The neutral losses in a spectrum are the mass differences between the molecular ion and other
major ions in the spectrum. Neutral-loss peaks can be very characteristic as spectral features for
certain classes of compounds.

In a neutral loss search, the algorithm examines the submitted spectrum and identifies the
molecular ion. It submits the mass value of the molecular ion to the search algorithm along
with the spectrum. The search algorithm calculates the significant neutral losses and compares
them with the library data. It returns hits according to matches of the molecular ion and its
neutral losses.
MS/MS Hybrid Applies an MS/MS hybrid similarity-search algorithm for library matching of spectra.

This option uses a combination of the simple and neutral-loss search strategies. In this search,
the algorithm searches for an MS/MS spectrum in a library of MS/MS spectra.
Options
Search With MW= Restricts the library search to entries with a particular molecular weight. Use the associated box
to enter the molecular weight.

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Table 50. NIST Search page parameters (Sheet 3 of 4)

Parameter Description
Reverse Search Sorts matching library spectra by the reverse search match factor. By default, the algorithm
sorts matches by the forward match factor.
Penalize Rare Limits the impact of rare compounds by reducing the match factor.
Compounds
Use this option only when you select one or more of the NIST databases (such as MAINLIB).
It has no effect on spectra in user libraries or other commercial libraries.

Each reference spectrum in a NIST library contains a record of other commercial databases
containing information about the compound. A compound is considered rare if it is present in
a limited number of these databases.

When you select the Penalize Rare Compounds option, the application reduces the match
factors for matched compounds that are present in few or no databases other than the NIST
libraries. The maximum reduction penalty is 50 out of 1000.

Selecting this option leads to a relative increase in the match factors of common compounds,
placing them higher in the match list than exotic isomers with nearly identical spectra.
Mass Defect

Use this area to set the parameters for library searches to correct for the differences between the actual masses and the
nominal integer masses of the atoms in a molecule. Assign a larger mass defect (in millimass units) for more massive
molecules because, in general, they are composed of more atoms than less massive molecules. More massive molecules
need a larger correction factor to approximate the linear function that the application uses to calculate masses.
Enable Include mass defect values for library searches.
Defect (mmu) Specify the mass defect (in millimass units).

Specify a smaller value for the lower mass ranges in the first box and a larger value for the
higher mass ranges in the second box.
At Mass (amu) Specify the masses at which the application applies the specified mass defect values to calculate
mass.

Specify a smaller mass value in the first box and a larger mass value in the second box.
MS/MS and In-source HiRes Search Options

Use this area to set the parameters for library searches involving the comparison of peaks in the search spectrum with
library spectra whose precursor m/z value or product mass spectral peak might need to fall within a specified tolerance
setting.
Precursor +– Specify the range of the m/z tolerance for precursor ions, in either ppm or m/z units.

Range: 0.015 to 100 000 ppm or m/z 6×10–5 to 500; default: 10 ppm
Product Ions +– Specify the range of mass spectral peak tolerance for product ions, in either ppm or m/z units.

Range: 0.015 to 100 000 ppm or m/z 6×10–5 to 500; default: 10 ppm

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Table 50. NIST Search page parameters (Sheet 4 of 4)

Parameter Description
Button
Make Default Saves the current settings as the default settings.
Apply Starts the library search with the entered settings.
Help Opens the FreeStyle Help to the NIST library search topic.

Modifying an mzVault Search from the mzVault Search Page

Use the mzVault Search page in the Info Bar to search the mzVault library for library entries
that match your selected fragmentation spectrum. You can filter the search by compound
name, chemical formula, or precursor m/z value.

Follow these procedures:

• To open the mzVault Search page
• To modify the display in the mzVault Search Results view
• To reset the mzVault search parameters and query spectrum

Y To open the mzVault Search page

Run an mzVault search (see Performing a Local NIST or mzVault Library Search).
When the search is complete, you can reset the search parameters.
Figure 146 shows the mzVault Search page.

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Figure 146. mzVault Search page

Y To modify the display in the mzVault Search Results view

In the Display list on the mzVault Search page, make a different selection (see Display in
Table 51).

Y To reset the mzVault search parameters and query spectrum

1. When applicable, click the Browse icon next to the Library list and select another
mzVault library.

Note The FreeStyle application lets you select the access-restricted mzVault library.

2. In the Hits box, type the maximum number of compounds that you want the search to
return.
3. From the Display list, select how you want to display the matching spectra in the
Spectrum view (see Setting Up the Default Library Search Parameters).

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4. (Optional) In the Score Cutoff box in Search Parameters area, specify the minimum score
for a match to be returned.
Default: 0
5. Select the type of search to conduct, either Forward or Reverse.
6. (Optional) For filtered searches, do the following:
a. Select the check box on the left to make the filter selections available. You can select
up to six filters.
b. Select a filter type. You can select the filters from the 12 filter types from the
dropdown menu.
c. Specify the filter criterion for the selected filter.
For details about selecting the type of filters, see Table 51.
d. From the Match/Custom dropdown list, select Match to update the filter criteria
based on the selected scan, or Custom to retain the edited filter criteria as you
navigate to different scans. The Custom option is applicable only for the following
filter types:
• Collision energy
• Precursor m/z
• Retention time
• Retention time range
7. In the Fragment Tolerance area, select the Automatic or Manual option to indicate how
to specify the fragment tolerance.
8. (Optional) Select whether you want the application to filter out low intensity peaks from
the query spectrum before performing the search.
9. (Optional) To have the mzVault application ignore any mass that is within 2.2 Da of the
precursor ion for the purpose of calculating scores, select the Remove Precursor check
box.
10. Click Apply.
The application applies the search parameters and displays the mzVault search results for
the selected criteria.

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Table 51 describes the parameters for the mzVault Search page in the Info Bar.
Table 51. mzVault Search page parameters (Sheet 1 of 4)
Parameter Description
Search List

Use the settings in this area to choose the library for library searches. Specify how many search results to display and
how they are displayed.
Library Select the library (database file) for the search.
Hits Specify how many search results to display in the mzVault Search Results View.

Range: 1–10 000; default: 10

Display Select how to display the library spectrum and query spectrum:
• Stacked—Displays the query spectrum above the currently selected library spectrum in
the match list.
• Mirror—Plots the query spectrum on the positive y axis from 0 to 100% intensity and
the library spectrum on the negative y axis from 0 to –100% intensity.
• Exclusion—Subtracts the library spectrum from the query spectrum.
Search Parameters
Score Cut-Off Filters out results below a percentage of the highest score. For example, if the highest score
for a search result is 0.900 and the setting in this box is 60, the search does not return results
with scores below 0.540 (0.9 × 60% = 0.540).

Range: 0.00 to 100.00; default: 0.00

Search Type
Forward Conducts a forward search, which bases the search on a comparison of the peaks in the
unknown spectrum against the peaks in a library spectrum.

When the unknown spectrum includes a peak that is not in a given library spectrum, the
score for the match is negatively affected.

Use a forward search when the unknown spectrum is of high quality—that is, when it has
good fragmentation and few low-intensity background peaks.
Reverse Conducts a reverse search, which bases the search on a comparison of the peaks in a library
spectrum against the peaks in the unknown spectrum.

When the unknown spectrum does not contain a peak that is in the library spectrum, the
score for the match is negatively affected, but the presence of additional peaks in the
unknown spectrum has no effect on the score.

Use a reverse search if the unknown spectrum includes peaks from several components or
has a lot of background noise.

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Table 51. mzVault Search page parameters (Sheet 2 of 4)

Parameter Description
Filters
Collision Energy Filters the library entries with scan data acquired at the specified collision energy within the
specified tolerance.

Valid entries: Numeric value > 0

Compound Filters the library entries by the name or part of the name of the compound.

Valid entries: Alphanumeric and special characters

Compound Class Filters the library entries for the selected compound class.

The Compound Class list includes only compound classes found in the selected library.
Curation Type Filters the library spectra for the selected curation type.

The Curation Type list includes only the curation types in the selected library.
Formula Filters the library entries by the chemical formula of the library entry.

Valid entries: Alphanumeric and special characters

Fragmentation Mode Filters the library spectra with the same fragmentation mode as the query spectrum.

Default: Set to Match

Peptide Sequence Filters the library entries that include the specified peptide sequence.
Precursor mass range Filters the library spectra for the specified range of precursor m/z values.

Valid entries: The maximum value must be greater than the minimum value by 0.1 amu
Precursor m/z Filters the library entries by the mass-to-charge ratio of the precursor ion for the library
fragmentation scans. When you select this option, the Precursor m/z and the Precursor
Tolerance boxes become available.

Valid entries: Numeric values up to four decimal places.

Retention Time Filters the library spectra from the specified retention time within a tolerance.

Default: Retention time of the query scan; valid entries: Positive numeric values
Retention time range Filters the library spectra from the specified retention time range.

Valid entries: The maximum value must be greater than the minimum value by 0.1 min.
Scan Filter Filters the library spectra for the specified scan filter.

Valid entries: All or part of a valid scan filter; alphanumeric characters

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Table 51. mzVault Search page parameters (Sheet 3 of 4)

Parameter Description
Filter Criterion

Defines the filter criteria based on the Filter type.

Match/Custom
Match Updates the filter criteria based on the selected scan.
Custom Retains the edited filter criteria as you navigate to different scans.
Query Spectrum

Use the settings in this area to define the mass tolerance and Intensity threshold settings.
Fragment Tolerance
Automatic (Default) Uses an internal algorithm to calculate the mass tolerance.
Manual Selecting this option activates the mass tolerance box and the corresponding units list. Enter
the mass tolerance for the fragment ions.

Units: amu, mmu, or ppm

Range: 0.001 to 1000.00; default: 10.000

Intensity Threshold
On or Off Determines whether the application filters out peaks with an intensity below the Intensity
Threshold (%) setting.
• (Default) On—Filters out peaks that are less than the specified value. Makes the
Intensity Threshold% parameters available.
• Off—Does not filter out peaks on the basis of their intensity.

The Intensity Threshold parameter calculates the score that indicates how well a spectrum
matches a library spectrum and displays the score in the High Res Score column.
Intensity Threshold%
Automatic (Default) Uses an internal algorithm to calculate the threshold value.
Manual Lets you to specify the threshold value to use for filtering spectrum peaks. The application
takes the default value from the scan that you selected from the raw data file.
Additional Query Spectrum parameter
Remove Precursor Ion Determines whether the application removes peaks within 2.2 Da of the precursor ion. In
an MS/MS experiment, the fragmentation spectrum does not normally contain a peak for
the precursor ion, but sometimes it can appear and interfere with the scoring algorithm.
This option corrects for the presence of a peak for the precursor ion.

Default: Selected

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Searching the Online mzCloud Mass Spectral Database

Table 51. mzVault Search page parameters (Sheet 4 of 4)

Parameter Description
Button
Make Default Saves the current settings as the default settings.
Apply Starts the library search with the entered settings.
Help Opens the FreeStyle Help to the mzVault search topic.

Searching the Online mzCloud Mass Spectral Database

The mzCloud mass spectral database includes thousands of fragmentation spectra.

Y To perform an mzCloud library search for a matching fragmentation spectrum

1. In a Spectrum view, select a fragmentation spectrum.

2. In the Workspace Processing toolbar, choose Library Search > mzCloud Search.

Figure 147 shows the mzCloud Spectrum Search dialog box that opens in your default
browser when you run an mzCloud search.
Figure 147. mzCloud search parameters in a browser

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Exporting a Mass Spectrum to the NIST MS Search Application

Figure 148 shows the search results for an mzCloud search.

Figure 148. mzCloud search results

Exporting a Mass Spectrum to the NIST MS Search Application

You can export a mass spectrum to the Library Search page of the NIST application.

Note Thermo Fisher Scientific provides the NIST application with the Xcalibur data
system.

Y To export a spectrum to the NIST application

1. Click the Spectrum view or spectrum plot of interest.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.
3. In the Library Search area of the toolbar, click Export to NIST.
The Library Search page of the NIST application opens with the search spectrum
displayed.

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Managing Libraries

Managing Libraries
Use the Thermo Library Manager dialog box to add a library, copy a library, or delete a
library.

When you add a library to the NIST libraries list, either copy the library file to the local
computer or link to the library at a remote location without copying files.

Y To add a library

1. Click the Workspace Processing tab to open the Workspace Processing toolbar.
2. Click Library Manager to open the Thermo Library Manager dialog box (see
Figure 149).
Figure 149. Thermo Library Manager dialog box

3. Click Add to open the Add Library dialog box (see Figure 150).
Figure 150. Add Library dialog box

4. Type the path for the new library file in the Source box, or click Browse to locate the file.
5. Select one of two options:
• Copy the library to the local computer.
• Link to the library from either a remote location or computer.

Tip Because libraries can be large, select the link option rather than the copy option
to save time.

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Managing Libraries

6. Click OK.
The data system adds the library to the NIST Libraries list in the Thermo Library
Manager dialog box.
7. When you have no more tasks to complete with the Library Manager, click Exit.

Y To copy a library for archiving

1. In the Thermo Library Manager dialog box (see Figure 149), select a library in the NIST
Libraries list.
2. Click Archive.
The Archive Library dialog box opens (see Figure 151).
Figure 151. Archive Library dialog box

3. In the Archive Library dialog box, type the path for the copied library file in the
Destination box, or click Browse to find the location.
4. To copy the selected library to the remote location, click OK.
5. When you have no more tasks to complete with the Library Manager, click Exit.

You can copy a selected library to another directory on the computer or network by using the
Archive feature.

Y To delete a selected library in the NIST Libraries list

1. In the Thermo Library Manager dialog box (see Figure 149), select a library to delete
from the NIST Libraries list.
2. Click Delete.
The system prompts you to confirm the deletion.
3. Click Yes.
4. When you have no more tasks to complete with the Library Manager, click Exit.

IMPORTANT After you delete a library, you cannot bring it back. Make sure that you
want to delete a library permanently before you proceed, especially a shared library on the
network.

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Spectrum Workspace Processing Toolbar – Library Search Features

Spectrum Workspace Processing Toolbar – Library Search Features

Use the features in the Library Search area of the Workspace Processing toolbar to perform
library searches.

Y To display the Spectrum Workspace Processing toolbar

1. Click a Spectrum View or a MultiSpectrum View to select it.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.

Figure 152 shows the Library Search area in the Workspace Processing toolbar, and Table 52
describes the toolbar icons.
Figure 152. Library Search areas of the Workspace Processing toolbar

Library Search features

Table 52. Spectra-specific Workspace Processing icons

Command Description
Library Search area
Library Search menu
NIST Search Opens the Library Search page in the Info Bar, where you run a NIST library search on the
selected spectrum and displays the results in the NIST Search Results view. You can modify
the library search parameters on the Modifying a NIST Search from the NIST Search Page of
the Info Bar. See Performing a Local NIST or mzVault Library Search.
mzVault Search Opens the mzVault Search page in the Info Bar, runs an mzVault library search on the selected
spectrum, and displays the results in the mzVault Search Results and mzVault Chemical
Structure views.
mzCloud Search Uploads a spectrum to mzCloud.org for a search. The website opens and displays the search
parameters (see Figure 147). Select the appropriate settings and click OK. The website
displays the results (see Figure 148). See Searching the Online mzCloud Mass Spectral
Database.
Export to NIST Exports a spectrum to the NIST application for a search. The NIST application opens and
displays the search results. See Exporting a Mass Spectrum to the NIST MS Search
Application.
Library Manager Opens the Thermo Library Manager dialog box, where you select or create NIST libraries.
See Managing Libraries.

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 9

Simulating Isotope Distributions

The FreeStyle application includes a utility that predicts and displays the isotopic distribution
for a specified chemical formula or peptide sequence. The simulated spectrum is based on the
elemental composition of the compound or peptide and the charge state distribution of its
adduct ions.

To display simulated isotope distribution plots for chemical formulas and peptide sequences,
see these topics.

Contents
• Simulating the Isotope Distribution for a Chemical Formula or Peptide
• Displaying Centroid and Profile Spectra in the Same Window
• Isotope Simulation Page

Simulating the Isotope Distribution for a Chemical Formula or Peptide

A simulated isotopic distribution is based on a chemical formula or peptide sequence. For
details about the parameter settings, see Isotope Simulation Page.

When you open the FreeStyle application, the Isotope Simulation page opens in the Info Bar.
Without opening a raw data file, you can use the Isotope Simulation page to display, in a
separate window, the isotope pattern at a specified resolution for compounds or peptides.
After opening a raw data file (see To open a raw data file or a sequence file), you can also use
the Isotope Simulation page to insert a simulated isotope pattern or profile spectrum in a
Spectrum view and an EIC trace in the Chromatogram view of a Workspace.

Y To display isotope simulations

1. If the Isotope Simulation page is not visible, click the Isotope Simulation tab.
2. For the first simulation, do one of the following:
• To display the simulation in the Isotope Simulation window, keep the New option.
–or–

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Simulating the Isotope Distribution for a Chemical Formula or Peptide

• To display the simulation in the Spectrum view of a Workspace, open a raw data file,
and then on the Isotope Simulation page, select the Show within the Workspace
check box.
Figure 153 shows the default settings on the Isotope Simulation page.
Figure 153. Isotope Simulation page without an open workspace
Display options for the Isotope
Simulation window

Select to display the simulated

spectrum within the Workspace
instead of in a separate window.

3. Enter the formula for a compound or the sequence of a peptide:

• For a compound, select Chemical Formula and enter the chemical formula using the
chemical symbols in the periodic table.

Elemental composition of caffeine

• To specify a specific isotope, use square brackets before the element name to enclose
the isotope mass number, for example, [13]C.

Elemental composition of caffeine where one carbon-12

atom has been replaced with carbon-13

Elemental composition of caffeine where all the carbon-12

atoms have been replaced with carbon-13

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 9 Simulating Isotope Distributions
Simulating the Isotope Distribution for a Chemical Formula or Peptide

• To specify repeating moieties, such as those found in polymers, use parentheses.

Polyethylene glycol

• For a peptide, select Peptide and enter the peptide sequence using uppercase
one-letter symbols for the amino acid residues (see One- and Three-Letter
Abbreviations for Amino Acid Residues).

Kentsin, a tetrapeptide with the following amino acid

sequence: Threonine-Proline-Arginine-Lysine

Note If you enter an incorrect formula, the application outlines the Formula box in
red. Examples of incorrect entries include the following:
• Peptide—Lowercase letters or three-letter abbreviations
• Chemical formula—Chemical symbols that are not in the periodic table

4. Under Adduct and Charge Distribution, do the following:

a. Select the polarity of the ion.
b. Select the adduct species, the charge of the most abundant ion, the concentration of
the adduct species, and the half width of the charge distribution.
For example, to simulate a spectrum for both the protonated and sodiated adduct
ions of a compound in a high-concentration sodium solution, select the following:
• Polarity: positive
• Species: Na
• Concentration: High
• Half-Width: 0
5. To display an EIC trace for the specified ion in the Chromatogram view of a Workspace,
open a raw data file if you have not already done so, and then select the Show Extracted
Ion Chromatogram check box.
6. In the Simulated Spectrum area, do one of the following:
• To display a pattern, leave the Profile check box clear.
–or–
a. To display a simulated profile spectrum, select the Profile check box.
b. (Optional) Specify the resolution.

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Simulating the Isotope Distribution for a Chemical Formula or Peptide

Result:
• When you are working with raw data, the application automatically populates
the Resolution box with the experimental resolution in the current raw data file.
• When you have not opened a workspace, the application populates the
Resolution box with the default value of 1 000 000. You can type a value from 1
to 2 000 000.

Tip You can specify a simulated profile spectrum to determine whether your
instrument is capable of resolving the theoretical isotope pattern for a specific ion,
or you can specify several simulations to determine the required resolution.
c. Select the centroiding algorithm.

Algorithm Calculation
Apex Samples Uses the sampling points nearest the peak apex.
All Samples Uses all the sampling points in the profile peak, down to the
baseline or valley between the resolved peaks.

Figure 154 shows a portion of a simulated profile spectrum. The two graphs show the
difference between the two centroiding methods, where the left graph displays the
results of the Apex Samples algorithm and the right graph displays the results of the
All Samples algorithm. When you zoom in on the peak apexes, you can see the slight
difference between the centroiding methods.

Note When you display a simulated profile spectrum in the Spectrum view of a
Workspace, the application displays the centroids for the Gaussian-shaped peaks
as vertical green lines.

Figure 154. A1 peaks in a simulated profile spectrum (in the Spectrum view)
Apex Samples All Samples

Centroid

When you leave the Show within the Workspace check box clear, the Insert and Replace
options become available after you run the first simulation and the Isotope Simulation
window opens.

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 9 Simulating Isotope Distributions
Simulating the Isotope Distribution for a Chemical Formula or Peptide

7. Click Apply.
8. For additional simulations, do the following:
a. Do one of the following:
• To display additional simulations in separate Isotope Simulation windows, select
New.
• To insert additional simulations into one Isotope Simulation window, select
Insert.
–or–
• To replace the current simulation with another simulation, select Replace.
b. Modify the compound or peptide information, and then click Apply.

Figure 155 shows the result of an isotope simulation for the protonated ion of caffeine.
Figure 155. Simulated centroid spectrum and EIC trace shown within the Workspace

Extracted ion
chromatogram

Simulated
centroid
spectrum

Note Most peptides have multiple charge sites, which means that their spectra will show
multiple charge states.

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Simulating the Isotope Distribution for a Chemical Formula or Peptide

Figure 156 shows an isotope simulation for a peptide with a charge distribution pattern of
5,1—that is, a charge of +5 and a half width of 1. Notice that the monoisotopic peak (A0) is
not the most intense peak. In compounds with large numbers of carbon atoms, the
probability that one of the atoms will be a carbon-13 atom increases.
Figure 156. Example isotope simulation for a peptide

As shown in Figure 156 and Figure 157, when the number of carbon atom exceeds 91, the
probability that the ion includes one carbon-13 atom exceeds 100% and the M+1 peak for the
(A1) ion with one carbon-13 atom becomes the most intense peak.

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 9 Simulating Isotope Distributions
Displaying Centroid and Profile Spectra in the Same Window

Figure 157. m/z 692 isotope envelope

A1 peak with one carbon-13 atom

A0 monoisotopic peak

Displaying Centroid and Profile Spectra in the Same Window

The application does not display formula labels for simulated profile spectra. To view the
formulas for the simulated peaks, run the simulation in both the profile and centroid modes
within the same Isotope Simulation window.

Y To view both simulation types in the Isotope Simulation window

1. Specify the initial simulation, make sure that the Show Within the Workspace and Profile
check boxes are clear, and click Apply.
2. Select the Insert option.
3. Specify a second simulation for the same formula and charge distribution.

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Displaying Centroid and Profile Spectra in the Same Window

4. Select the Profile check box, and then select the centroiding algorithm.
5. Click Apply.
Figure 158 shows the isotope simulation and the mass spectrum for the protonated
caffeine ion. At the experimental resolution, the isotope simulation shows that the A1
peaks at m/z 196.1 will not be completely resolved. The experimental spectrum shows a
similar profile at m/z 196.1.
Figure 158. Simulation for caffeine versus the experimental spectrum for a caffeine sample

Expected mass peaks for the A1

isotopes for a centroid simulation

A1 peaks for a profile simulation

A1 peaks in the experimental spectrum

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 9 Simulating Isotope Distributions
Isotope Simulation Page

Isotope Simulation Page

Use the Isotope Simulation page of the Info Bar to create a simulated isotopic distribution
plot. The Isotope Simulation page is always available. Without opening a workspace, you can
only display a simulated spectrum in the stand-alone Isotope Simulation window. After you
open a workspace, you can display an EIC trace in the Chromatogram view and add an
isotope simulation plot to the Spectrum view.

For information about running the isotope simulation utility and reviewing the resulting
spectrum, see Simulating the Isotope Distribution for a Chemical Formula or Peptide.

Table 53 describes the parameters for the Isotope Simulation page.

Table 53. Isotope Simulation page parameters (Sheet 1 of 5)
Parameter Description
New Opens a separate Isotope Simulation window each time you click
Apply.
Insert Adds repeated simulations to the same Isotope Simulation window.

Available when an Isotope Simulation window is open.

Replace Replaces the current simulated spectrum with the latest simulation.

Available when an Isotope Simulation window is open.

Type Specify that the formula is either a Chemical Formula or a Peptide.

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Isotope Simulation Page

Table 53. Isotope Simulation page parameters (Sheet 2 of 5)

Parameter Description
Formula Enter the formula or peptide sequence for the compound or peptide
for which you want to create a simulated spectrum.

After you enter a formula or peptide sequence, it appears in a selection

list for this box.
• Chemical Uses the custom periodic table elements for isotope simulation when
Formula you select the custom periodic table for elemental composition.

For entering a chemical formula, do the following:

• Use the IUPAC symbol for each element—that is, match the
notation, including the capitalization, in the periodic table of
elements.
• To enter a specific isotope, use square brackets before the element
name to enclose the isotope mass number, for example, [13]C.
• Use parentheses to specify repeating moieties such as those found
in polymers, for example, HO(C2H4O)5H.
Note To create a custom adduct, enter any valid chemical formula,
select a species, and type or select the charge of the adduct ion.
Note To specify mixtures of substances, use + (addition) and
* (multiplication) symbols. Both are required to specify a mixture. A
valid mixture has the format substance*quantity + substance*
quantity, for example, C4H8*2+H2O*5.
• Peptide For a peptide sequence, do the following:
• Use the single capital letter abbreviations for amino acids.
• You can specify mixtures of substances by using additional
symbols “+” (addition) and “×” (multiplication). Both symbols are
required to specify a mixture. A valid mixture has the format
substance × quantity + substance × quantity, for example,
A × 2 + C × 5.

See One- and Three-Letter Abbreviations for Amino Acid Residues.

The maximum molecular weight for the formula is less than
600 000 amu.
Adduct and Charge Distribution
Polarity Select the Positive or Negative option.

Default: Positive

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Isotope Simulation Page

Table 53. Isotope Simulation page parameters (Sheet 3 of 5)

Parameter Description
Species Select, if necessary, the adduct for the simulated spectrum.
• For positive polarity, you can select H, K, Na, NH4, or no adduct.
Or, you can type the chemical symbol or formula for a positive
adduct such as Ca for calcium.
• For negative polarity, you can select H, Cl, OH, HCOO, or no
adduct. Or, you can type the chemical symbol or formula for a
negative adduct.
Charge Specify the charge of the adduct ion or peptide.
• (Positive polarity) Range: 1–25; default: 1
• (Negative polarity) Range: –1 to –25; default: –1
Half Width Simulate the number of additional charges on each side of the most
abundant ion. Select a value from 0 to 99.

For example: When you simulate charge 5 and a half width of 2, then
the data system draws charges 5 ± 2, giving 7, 6, 5, 4, 3 (with the
largest mass peak (normalized to 100% relative intensity) at charge 5).

For most small molecules, simulate charge 1 with a half width of 0.

To simulate an intensity distribution, the peaks at the edge of the

distribution are shown at 5% of the height of the most abundant peak.

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Isotope Simulation Page

Table 53. Isotope Simulation page parameters (Sheet 4 of 5)

Parameter Description
Concentration There are four possibilities for how the adduct is added to the ion:
One, Low, High, and 100%.
• One adduct For positive charge states

The application creates the specified charge state by adding the

specified species for the first charge and H+ ions for subsequent
charges. For example, a charge distribution of Charge 2+ (most
abundant) and Half Width = 1 (2,1) and a Na+ adduct shows these
ions and the simulated spectrum includes three isotope clusters:
– Charge +1) Chemical_FormulaNa+
– Charge +2) Chemical_FormulaHNa+2
– Charge +3) Chemical_FormulaH2Na+3

For negative charge states

When you select H as the species, each charge state shows a loss of H+
to attain the specified charge—that is, a loss of one hydrogen ion for a
charge of negative one, a loss of two hydrogen ions for a charge of
negative two, and so on. When the molecule does not contain
hydrogen, nothing is removed.

For the neutral compound C8H8:

– Charge –1) C8H7
– Charge -2) C8H6
– Charge -3) C8H5

For negative species, such as Cl–, the –1 charge state shows the
addition of the negative species to attain the specified charge. Higher
charge states show the addition of one Cl– and the loss of one or more
H+ to attain the specified charge.
For the neutral compound C8H8:
– Charge –1) C8H8Cl
– Charge -2) C8H7Cl
– Charge -3) C8H6Cl
• Low The ion with no adduct is included at 100% intensity, one adduct at
25% intensity, two adducts at 11% intensity, and so on (to the limit of
the charge distribution).
• High The ion with N adducts are included at 100% intensity, N–1 adduct
at 25% intensity, N–2 adducts at 11% intensity, and so on, where N is
the absolute value of the maximum charge simulated (to the limit of
the charge distribution).

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Isotope Simulation Page

Table 53. Isotope Simulation page parameters (Sheet 5 of 5)

Parameter Description
• 100% The ion of charge N contains M adducts (where M is the absolute
value of N).
Show Extracted Displays an EIC trace for the monoisotopic ion in the Chromatogram
Ion view.
Chromatogram
Show within the Displays the isotope simulation in the Spectrum view of a Workspace.
Workspace
Simulated Spectrum
Profile and When the Profile check box is clear, the application generates a
Resolution theoretical isotope pattern with infinite resolution—that is, it displays
the peaks as one-dimensional sticks rather than as Gaussian peaks
similar to the intensity versus frequency data that the instrument
generates.

When the Profile check box is selected, for each peak in the simulated
pattern, the application uses a Gaussian peak-shape model that bases
the peak width on the specified resolution. The application then sums
the simulated Gaussian peaks to create a simulated profile spectrum
for the specified resolution. The application displays the centroid for
each profile peak as a green vertical line.

When you open a raw data file in the workspace, the Resolution box
displays the resolution value for the most intense peak (full width at
half maximum [FWHM]) in the selected scan. You can overwrite this
value.
Tip To view the resolution values for each mass spectrum peak, click
Peak Resolution in the Spectrum – Display Options toolbar.
Centroid • (Default) Apex Samples—Selects the sampling points nearest the
peak apex to calculate the centroid of each simulated profile peak.
• All Samples—Uses all the sampling points across the profile peak,
down to the baseline or valley between resolved peaks.
Button
Apply Calculates the isotope distribution and displays the simulated
spectrum by using the parameter settings.
Help Opens the FreeStyle Help to the isotope simulation topic.

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Isotope Simulation Page

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 10

Working with CV Plots for FAIMS Data

The Thermo Scientific high-field asymmetric waveform ion mobility spectrometry (FAIMS)
interface increases sensitivity by reducing chemical noise and matrix interferences. Use the
CV Plot view to determine the optimal compensation voltages for your FAIMS-MS
experiments.

Follow the procedures in these topics.

Contents
• CV Traces Dialog Box
• CV Plot View
• Export CV Data

CV Traces Dialog Box

The CV Traces dialog box includes check boxes for the selected traces in the Chromatogram
Ranges view.

Y To select the traces for display in the CV Plot view

1. Open an Xcalibur raw data file with data acquired from a mass spectrometer with the
FAIMS interface.
2. In the Chromatogram Ranges view, specify the traces of interest.
3. In the Workspace Options toolbar, click CV Plot.
The CV Traces dialog box opens (Figure 159).
4. Select the check boxes for the traces of interest.
5. Click OK.
The CV Plot view opens. For information about working with the CV Plot, see the next
topic CV Plot View.

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10 Working with CV Plots for FAIMS Data
CV Plot View

Figure 159. CV Traces dialog box

CV Plot View
Use the CV Plot view to determine the optimum compensation voltages for your
LC/MS/FAIMS experiments.

To open the CV Plot view, see CV Traces Dialog Box. You can reopen the CV Traces dialog
box to add or remove traces from this view.

Figure 160 shows a CV Plot view with three overlaid traces: a TIC MS trace in black, a mass
range m/z 507.2 ± 0.5 trace in red, and a mass range m/z 997.3 ± 0.5 trace in green.

The Filter page on the left shows scans acquired at 12 stepped compensation voltages from
0.00 to 1000 and two mass ranges. The CV Plot view, which plots the relative abundance
versus the compensation voltage, shows a maximum response at a compensation voltage of 20
for both mass ranges.

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 10 Working with CV Plots for FAIMS Data
CV Plot View

Figure 160. Workspace showing a selected CV Plot view

The CV Plot view, Chromatogram view, and Spectrum view are interactive—that is, as you
move the red vertical bar across the CV voltage range on the x axis, the traces update in the
Chromatogram and Spectrum views. Use this interactive behavior to determine the optimum
compensation voltage for your analytes—that is, monitor the relative abundance of the ions in
the Spectrum view as you move across the CV range in the CV Plot view.

At a CV of 10, the ion at m/z 508.00 is relatively more abundant than the ion at m/z 997.33
(Figure 161).

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CV Plot View

Figure 161. Compensation voltage 10

At a CV of 40, the ion at m/z 997.33 is relatively more abundant than the ion at m/z 508.00
(Figure 162).
Figure 162. Compensation voltage 40

CV label

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 10 Working with CV Plots for FAIMS Data
Export CV Data

The display options for the CV Plot view are similar to those for the Chromatogram view (see
“Chromatogram – Display Options Toolbar” on page 109). Use the CV Plot icon in the
Workspace Options toolbar to add the compensation voltage label to the y-axis maxima in the
CV Plot view.

Export CV Data
Use the Export CV Optimization Data command to export a single CSV file with merged
data from multiple CV plots.

Y To export CV data to a file

1. Use the Auto Filter dialog box to merge the CV filters.

See To specify the maximum number of chromatogram traces to display.
2. Right-click the CV Plot view and choose Export CV Optimization Data from the
shortcut menu.
3. In the Export Data dialog box, type a name for the CSV file and save.
The application saves the compound name, precursor mass, and FAIMS CV value (the
value at which the relative abundance is the highest) to an CSV file.

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Export CV Data

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 11

Peptide Fragment Annotation

The Thermo Scientific mass spectrometers support different strategies for acquiring peptide
fragment spectra. The ETD or CID activation types performed in mass spectrometers provide
different fragment scans of the same peptide.

This chapter explains how Peptide Fragment annotation in the Freestyle application annotates
the mass scans of the selected peptide sequence.

Contents
• Overview of Peptide Fragments
• Fragmentation Methods
• Annotating Peptide Fragments
• Peptide Fragments Info Bar
• Peptide Results View

Overview of Peptide Fragments

The fragment ions of peptides are produced by a CID or ETD activation process in which a
peptide ion is fragmented in a collision cell. The fragment ion spectra contain peaks of the
fragment ions formed by cleavage of the peptide bond and are used to determine the amino
acid sequence. A fragment must have at least one charge for it to be detected. When this
charge is retained on the N-terminal fragment, the ion is classed as a, b, or c. When the charge
is retained on the C-terminal fragment, the ion type is x, y, or z. A subscript indicates the
number of residues in the fragment.

The Peptide Fragment annotation displays all the fragmented ion series in the spectrum. The
fragment ion series includes a&x, c&z, and b&y.

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Fragmentation Methods

Fragmentation Methods
The Peptide Fragmentation in the Freestyle application helps you to annotate the fragment
ions for user defined peptide sequence in the spectrum. The spectrum header displays the
following details: name of the raw file, activation type (CID or ETD) used in fragmentation,
collision energy, and if the peptide is intact or fragmented. The peptide sequence is
fragmented in one of the following fragmentation methods:

CID—Uses the collision-induced dissociation method of fragmentation, where molecular

ions are accelerated to high kinetic energy in the vacuum of a mass spectrometer and then
allowed to collide with neutral gas molecules such as helium, nitrogen, or argon. The collision
breaks the bonds and fragments the molecular ions into smaller pieces. CID produces b and y
ions.

ETD—Uses the electron transfer dissociation method of fragmentation, where singly charged
reagent anions transfer an electron to multiply protonated peptides within an ion trap mass
analyzer to induce fragmentation. ETD cleaves along the peptide backbone while side chains
and modifications, such as phosphorylation, are left intact. This method is used to fragment
peptides and proteins. ETD produces primarily c and z ions.

The fragment ions produced are identified according to where they are fragmented in the
peptide. The a, b, and c fragment ions have a charge on the N-terminal side, and x, y, and z
fragment ions have a charge on the C-terminal side. Fragment ions a*, b*, and y* are ions that
have lost ammonia (–17 Da), and fragment ions ao, bo, and co are ions that have lost water (–
18 Da). The subscript next to the letter indicates the number of residues in the fragment ion.

Annotating Peptide Fragments

The spectrum view shows the annotated spectra of the selected peptide sequence. When you
use a CID or ETD fragmented peptide spectra, the view shows peaks of fragmented ions
formed by the cleavage of peptide bonds. These fragments are used to confirm the amino-acid
sequence.

Each alphabet represents an amino acid in the peptide sequence; for example, R for Arginine,
H for histidine, G for Glycine, and so on. The carboxyl group of one amino acid links to the
amine group of another amino acid to form an amino-acid sequence.

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 11 Peptide Fragment Annotation
Peptide Fragments Info Bar

Figure 163. Workspace Processing – Peptide Fragments

Peptide Fragments

Y To apply an amino acid sequence to a fragmented spectra

1. Open a raw data file.

2. Click the Spectrum view to make it active.
3. Click the Workspace Processing tab to open the Workspace Processing toolbar.
4. In the Protein Analysis area, click Peptide Fragments.
The Peptide Fragments page opens in the Info Bar.
5. Specify the parameter settings that you want to apply.
Table 54 describes the parameters for the Peptide Fragments page.
6. Click Apply.
The application runs and annotates the selected fragment spectra.

Peptide Fragments Info Bar

Use the Peptide Fragments page in the Info Bar to specify the parameter settings.

Y To display the Peptide Fragments page

1. Select the Spectrum view to make it active.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.
3. Click Peptide Fragments.

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Peptide Fragments Info Bar

Figure 164. Peptide Fragments Info Bar

Table 54 describes the parameters for the Peptide Fragments page.

Table 54. Peptide Fragments Page
Parameter Description
Peptide Fragments
Amino Acid Sequence Specifies the amino acid sequence that you want to use to
annotate the spectrum.
Charge Specifies the charge state of the precursor ion. You can either select
or manually enter a value.
Mass Tolerance Specifies the mass tolerance. The application annotates the
spectrum with ions within this mass tolerance. You can use
daltons (Da) or millimass units (mmu).
Activation Type
CID Collision Induced Dissociation. The application automatically
selects the option based on the spectrum selected.

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Peptide Results View

Table 54. Peptide Fragments Page

Parameter Description
ETD Electron Transfer Dissociation. The application automatically
selects the option based on the spectrum selected.
IMPORTANT By default, the FreeStyle application selects CID
as the activation type for all spectrum types other ETD (for
example, HCD, PQD, and so on).
Display Options
Ion Series Specifies the types of ion series to annotate the spectra. The
application automatically selects the b and y ion series for CID
activation type and c and z ion series for ETD activation type.
Charge State Displays the charge state of the precursor ion as stored in the scan
header of the raw data file. You can change the charge state to
filter the fragment ions.
Show peptide results Select or clear to show or hide the Peptide Fragments table.
table

Peptide Results View

The Peptide Results table shows the analyzed spectra of the selected peptide sequence. It
displays the major fragment ion series in the spectrum. The fragment ion series includes a, b,
c, x, y, and z ions, as well as water and ammonia losses from b and y fragment ions. You can
use the Peptide Results table to identify the spectral peaks that matches the amino-acid
sequence provided.

Y To display or hide the Peptide Fragments Table

1. In the Peptide Fragments page of the Info bar, select the Show Peptide Results Table
check box.
The Peptide Fragments table appears.
Figure 165. Peptide Fragments Table

2. To hide the table, clear the check box.

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Peptide Results View

The header of the Peptide Fragments table displays the terminal amino acid(s) or N-terminus
for each ion; for example, (NH3+) - A>. Each entry in the grid displays the m/z value of the
indicated ion.

The Spectrum view displays vertical and horizontal color-coded lines to indicate the position
of amino acid ions. The vertical color-coded bars indicate the position of matched ions and
horizontal lines indicate the amino acids matched between adjacent ions. Each horizontal line
also displays the ion series letter code and selected charge state.
Figure 166. Spectrum view
Letter code and
charge state

The following table describes the fragment ion series used in the activation type to annotate
the spectra.
Table 55. Fragment ion series (Sheet 1 of 2)
Activation type Ion series Description
CID b b ion with charge on the
N-terminal side
CID b* b ion that has lost ammonia
(-17 Da)
CID bo b ion that has lost water (-18
Da)
CID y y ion with charge on the
C-terminal side

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Filtering the Fragmented Ions

Table 55. Fragment ion series (Sheet 2 of 2)

Activation type Ion series Description
CID y* y ion that has lost ammonia
(-17 Da)
CID yo y ion that has lost water (-18
Da)
CID Immonium Immonium ion
ETD a• a ion
ETD z• z ion
ETD c´ c ion
ETD y´ y ion

In Peptide Fragments table, colors have the following significance:

• White indicates that the peptide was not found.
• Yellow represents a series fragment ions.
• Gold represents b series fragment ions.
• Blue represents y series fragment ions.
• Orange represents c series fragment ions.
• Green represents z series fragment ions.

By default, the Peptide Fragments table displays all ion types searched in the analysis. You can
remove any of these by clearing the appropriate ion check box on the right.

Filtering the Fragmented Ions

After you apply the parameters given in the Peptide Fragments Info Bar page, the Peptide
Fragments table and Spectrum view displays all ion types based on the parameters defined.
You can use the Display Options to filter the data.

Y To apply filters in the display options

1. In the Ion Series area, select or clear the check box for the appropriate ions in the Ion
Series area. In CID activation type, b and y ions are selected by default. In ETD
activation type, c and z ions are displayed by default.
2. In the Charge State box, select All or specify a charge state from the dropdown to filter
the charge states.
The Spectrum view and Peptide Fragments table display changes based on the filters
selected.

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Filtering the Fragmented Ions

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 12

Viewing Experiment and Instrument Information

In addition to containing the acquired data, the raw data file contains information about the
scan header, instrument status, instrument method, tune method, and sample. Use the
FreeStyle application to extract this information from the raw data files. Viewing the
experiment information for a particular acquisition can help you interpret the results of that
acquisition. Viewing the mass spectrometer readback (MS Trending detector type)
information for the acquisition can help you troubleshoot anomalies in the data.

To review the experiment and instrument information in your raw data files, follow the
procedures in these topics.

Contents
• Workspace Options Toolbar – Report Area
• Spectrum List – Display Options Toolbar
• Scan Header View
• Status Log View
• Instrument Method View
• Tune Method View
• Sample Information View
• File Header View
• Error Log View

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Workspace Options Toolbar – Report Area

Workspace Options Toolbar – Report Area

Use the commands in the Report area of the Workspace Options toolbar to view information
that is contained in the raw data file.

Y To display the Workspace Options toolbar

Click the Workspace Options tab.

Figure 167 shows the Report area, and Table 56 describes the menu commands, which display
information-specific views.
Figure 167. Report commands

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 12 Viewing Experiment and Instrument Information
Spectrum List – Display Options Toolbar

Spectrum List – Display Options Toolbar

Table 56. Workspace Options toolbar – Report area commands
Command Description
Spectrum List Displays the Spectrum List View, showing spectral peak information in a table.

Click the chromatogram trace to display the spectrum list for the chosen retention time and
scan number.
File Header Displays the File Header View.
Scan Header Displays the Scan Header View, showing the scan header of the active raw data file.

Click the chromatogram trace to display the scan header for the chosen retention time and
scan number.
Instrument Method Displays the Instrument Method View, showing the instrument method parameters that the
instrument used to obtain the raw data file.
Status Log Displays the Status Log View, showing mass spectrometer readbacks.

Click the chromatogram trace to display the status log for the chosen retention time and scan
number.
Tune Method Displays the Tune Method View, showing the tune parameters that the instrument used to
obtain the raw data file.
Sample Information Displays the Sample Information View, showing sample-specific information.
Error Log Displays the Error Log View, showing errors recorded during data acquisition.

Use the Exception Flags icon in the Spectrum List – Display Options toolbar to add or
remove the Flags column.

Y To display the Spectrum List – Display Options toolbar

1. Click the Spectrum List view to select it.

2. Click the Display Options tab to open the Display Options toolbar.

Figure 168 shows the Spectrum List – Display Options toolbar.

Figure 168. Spectrum List Display Options toolbar

The Exception Flags icon is the only icon in the Spectrum List – Display Options toolbar. It
displays or hides the Flags column in the Spectrum List view.

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Scan Header View

Scan Header View

Use the Scan Header view to display scan parameters and instrument data for the retention
time and scan number that you select in the Chromatogram view.

Y To open the Scan Header view

1. Click the Chromatogram view to select it.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Reports and choose Scan Header from the dropdown menu.

Figure 169 shows an example of a Scan Header view.

Figure 169. Scan Header view

The Scan Header view can include the following information:

• Scan parameters
• Instrument data
• Mass calibration parameters
• Ion optics readbacks
• Diagnostic data

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 12 Viewing Experiment and Instrument Information
Spectrum List View

Spectrum List View

The Spectrum List view lists in tabular form the peak positions, intensities, and relative
intensities of the peaks in the Spectrum view for the retention time that you select in a
Chromatogram view.

Y To open the Spectrum List view

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Reports and choose Spectrum List from the dropdown menu.

Figure 170 shows an example of a Spectrum List view and the associated mass spectrum.
Figure 170. Spectrum List view (bottom) associated with the mass spectrum (top)

Some raw data files contain flags that provide supplemental information about the peak data.
The possible flags are as follows:
• S—Saturated peaks are peaks with a signal too large to measure—that is, the signal was so
high that it was outside the dynamic range of the detector, causing saturation.
• R—Reference peaks are peaks from a reference compound used for an internal
recalibration of a scan.
• L—Lock peaks are local references used to calculate the accurate mass of nearby peaks.
• E—Exception peaks are peaks from a reference compound that are not used for
recalibration. These are typically small isotopes or fragments of the main references.

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Spectrum List View

• #—Mathematically modified peaks are peaks where the peak mass was recalculated by the
instrument, usually due to a calibration process.
• M—Merged peaks are peaks where the centroider combined two nearby peaks.
• F—Fragmented peaks are peaks separated into multiple peaks by the centroiding activity.
• T—Top peak in the cluster identified by the Advanced Peak Determination algorithm.
• U—Peak identified as an unresolved isotope where the resolution settings used to acquire
the data are not sufficient to resolve isotopes of a given mass and charge state as
determined from charge state deconvolution.

If the Spectrum List view does not show the Flags column (Figure 170), you can click
Exception Flags in the Display Options toolbar for the Spectrum List view to see this column
and the letters representing the flags.

Y To display flags in the spectrum list

Do one of the following:

a. Click the Spectrum List view to select it.
b. Click the Display Options tab to open the Display Options toolbar.
c. Click Exception Flags.
The Flags column appears as the last column. To hide the Flags column, click
Exception Flags again.
–or–
a. Click the Field Chooser icon, , to the left of the first column heading.
b. In the Field Chooser dialog box, select the Flags check box.

Y To choose columns for the spectrum list

1. Click the Field Chooser icon, , to the left of the first column heading.
The Field Chooser displays all available columns for the Spectrum List view.

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 12 Viewing Experiment and Instrument Information
Status Log View

Figure 171. Spectrum List Field Chooser

Values for the Average Mass column are available

instead of the values for the Monoisotopic Mass
column if the species is not isotopically resolved.

2. Select the check box for each column that you want to display.
The application immediately applies the column selections.

Status Log View

The Status Log view displays the mass spectrometer component readbacks that the system
recorded at the retention time selected in the Chromatogram view.

Y To open the Status Log view

1. Click the Chromatogram view to select it.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Reports and choose Status Log from the dropdown menu.

Figure 172 shows an example of the Status Log view. The view’s title bar includes the scan
number that corresponds to the selected retention time.

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Instrument Method View

Figure 172. Status Log view

You can display individual instrument status readbacks, as a function of the retention time, in
the Chromatogram view (see Setting up Instrument Status Traces). The readback display can
aid in troubleshooting anomalies in the data.

Instrument Method View

The Instrument Method view displays the instrument method. An instrument method is a set
of experiment parameters that define operating settings for the autosampler, liquid
chromatograph (LC), mass spectrometer, divert valve, syringe pump, and so on. Instrument
methods are saved as file type .meth.

For an LC/MS system, the Instrument Method view displays the instrument method settings
for the autosampler, LC pump, and mass spectrometer on separate pages. This information
does not change from scan to scan.

Y To open the Instrument Method view

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Reports and choose Instrument Method from the dropdown menu.
For example, the Autosampler page lists the injection-cycle information.
Figure 173 shows an example of the Autosampler page of an Instrument Method view.

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 12 Viewing Experiment and Instrument Information
Instrument Method View

Figure 173. Autosampler page of the Instrument Method view

Click these links to see more information
about these instruments.

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Instrument Method View

The LC page can include the solvent composition and the gradient information for the
acquisition.

Figure 174 shows an example of the LC page of an Instrument Method view.

Figure 174. LC page of the Instrument Method view

The Mass Spectrometer page might list the tune file, the lock masses, the scan event
descriptions, the activation types, the syringe pump settings, and the divert valve settings.

Figure 175 shows an example of the Mass Spectrometer page of the Instrument Method view.

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 12 Viewing Experiment and Instrument Information
Tune Method View

Figure 175. Mass Spectrometer page of the Instrument Method view

Tune Method View

The Tune Method view displays the settings in the tune method. A tune method is a defined
set of mass spectrometer tune parameters for the ion source, ion optics, and mass analyzer.

Y To open the Tune Method view

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Reports and choose Tune Method from the dropdown menu.

Figure 176 shows an example of a Tune Method view.

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Tune Method View

Figure 176. Tune Method view

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 12 Viewing Experiment and Instrument Information
Sample Information View

Sample Information View

Use the Sample Information view to view sample-specific autosampler, LC pump, and mass
spectrometer information in a raw data file. This information does not change from scan to
scan.

Y To open the Sample Information view

1. Click the Workspace Options tab to open the Workspace Options toolbar.
2. Click Reports and choose Sample Information from the dropdown menu.

Figure 177 shows an example of a Sample Information view.

Figure 177. Sample Information view

File Header View

Use the File Header view to view acquisition information about the current raw data file. The
File Header view displays information from the acquisition sequence, the autosampler, and
the mass spectrometer.

Y To open the File Header view

1. Open a raw data file.

2. In the Workspace Options toolbar, click File Header.
The File Header view opens above the other views.

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 Figure 178. File Header view

The view has three columns, each displays a custom label name and value as configured in the
Heading Editor dialog box.

Editing the File Header Values

Use the Heading Editor dialog box to edit the values in the File Header view.

Y To open the Heading Editor

1. Right-click the File Header, and click Heading Editor.

The Heading Editor dialog box opens.

Figure 179. Heading Editor dialog box

2. In the Heading Editor dialog box, choose a label name and value from the dropdown list
for each column.
3. Click Apply.
The selected values are populated in the File Header.
4. Click OK.

Table 57 describes the information in the Heading Editor dialog box.

Table 57. Heading Editor (Sheet 1 of 3)
Read-only parameter Description
The following values are from the sequence row that corresponds to the raw data file.
Acquisition Date/Time Displays the date and time that the displayed data was acquired.
Sample Name Displays the text in the Sample Name column.
Comment Displays the text in the Comment column.
Seq Row Displays the number of the sequence row.
 12 Viewing Experiment and Instrument Information
File Header View

Table 57. Heading Editor (Sheet 2 of 3)

Read-only parameter Description
Sample Type Displays the sample type selection in the Sample Type column.
Path Displays the directory and file name in the Path column, which is
the file’s original storage location and file name.
Cal Level Displays the calibration level in the Level column for calibration
standards.
Injection Volume Displays the injection volume, in microliters, in the Inj. Vol.
column.
Sample Weight Displays the numeric value in the Sample Wt column. The
application does not use this value for any internal calculations.
Sample Volume Displays the numeric value in the Sample Vol column. The
application does not use this value for any internal calculations.
Sample ID Displays the sample identification text in the Sample ID column.
ISTD Amount Displays the numeric value in the ISTD Corr Amt column.
CD Factor Displays the numeric value in the Dil Factor column. The
application does not use this value for any internal calculations.
Bar Code Displays the bar code information acquired by the autosampler’s
bar code reader during an injection sequence.
Bar Code Status Indicates whether the autosampler read the bar code for an
injection vial.
Inst Method Displays the instrument method in the Inst Meth column.
Proc Method Displays the processing method in the Proc Meth column.
User Text 1 to User Displays the text in the respective sequence table columns.
Text 5
An Xcalibur sequence table can include up to five additional text
columns for user-specific information. The default headings for
these columns are (1) Study, (2) Client, (3) Laboratory, (4)
Company, and (5) Phone. You can customize the column
headings in the Xcalibur data system’s User Labels dialog box.
The following values are from the autosampler. Not all autosamplers supply this
information to the data system.
Tray Index Displays text that identifies the autosampler tray.
Tray Name Displays text that identifies the type of autosampler tray.
Tray Shape Displays the shape of the autosampler tray (for example,
rectangular).
Vial Index Displays a numeric value.
Vials Per Tray Displays a numeric value.

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Error Log View

Table 57. Heading Editor (Sheet 3 of 3)

Read-only parameter Description
Vials Per Tray X Displays the number of vials across the autosampler tray.
Vials Per Tray Y Displays the number of vials that the autosampler is deep.
Instrument Name Displays the name of the mass spectrometer series (for example,
Orbitrap Fusion).
Instrument Model Displays the model name of the mass spectrometer (for example,
Orbitrap Fusion).
Instrument Number Displays the serial number of the mass spectrometer.
Instrument Software Displays the version number of the mass spectrometer’s
Version instrument control software that is installed on the data system
computer.
Instrument Hardware Displays the version number of the hardware components that are
Version installed on the mass spectrometer.
Flags Displays additional information about the scan data.
Mass Tolerance Displays the default mass tolerance setting on the Mass Options
page of the Xcalibur Configuration dialog box. The FreeStyle
application does not use this setting.
Tip To open the Xcalibur Configuration dialog box, from the
Xcalibur RoadMap view, choose Tools > Configuration.
Created by Displays the user name.
File Name Displays the name and path of the file storing the displayed data.

Error Log View

Use the error log to view errors recorded during data acquisition.

Y To open the Error Log view

1. Open a raw data file.

2. Click the Workspace Options tab to open the Workspace Options toolbar.
3. Click Reports and choose Error Log from the dropdown menu.
Figure 180. Error Log view

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 13

Running the Xtract Algorithm on Spectra and

Chromatogram Data
The Xtract algorithm of the FreeStyle application uses a fitting scheme to deconvolve and
deisotope isotopically resolved mass spectra of peptides, proteins, and nucleotides.

To deisotope and deconvolve the spectra for peptides, proteins, and nucleotides, follow the
procedures in these topics.

Contents
• Understanding the Xtract Algorithm
• Applying the Xtract Algorithm to an Isotopically Resolved Spectrum
• Xtract Page for a Selected Spectrum
• Deconvolved Spectrum View
• Xtract Results View
• Applying the Xtract Algorithm to a Chromatogram
• Xtract Page for a Selected Chromatogram

Understanding the Xtract Algorithm

The Xtract algorithm first examines a cluster of isotopically resolved peaks and uses the peak
spacing of a cluster to determine an initial estimate of the mass of the relevant component.
For peptides and proteins, it fits an averagine2 distribution to the observed peak profile in that
cluster to determine the monoisotopic mass that best reproduces that profile. (The
monoisotopic mass is the mass of an ion for a given molecular formula, which is calculated by
using the exact mass of the most abundant isotope of each element, for example,
C = 12.000000, H = 1.007825, and O = 15.994915.)

2
Senko, M.W.; Beu, S.C.; McLafferty, F.W. Determination of monoisotopic masses and ion populations for large
biomolecules from resolved isotopic distributions. J. Am. Soc. Mass Spectrometry. 1995, Vol. 6, 226–233.

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Applying the Xtract Algorithm to an Isotopically Resolved Spectrum

For nucleotides, the algorithm fits a distribution that is typical for nucleotides. Finally, the
Xtract algorithm combines the results for all observed charge states for each mass component
to produce a single mass value for that component. The resulting spectrum shows only the
monoisotopic masses and their ion populations for the components that the algorithm
identified.

When used properly, the Xtract algorithm reduces spectral noise and provides a high-intensity
mass spectrum of the monoisotopic peaks. You can use the Xtract Results table, which
contains the monoisotopic mass list of the deconvolved mass-spectral peaks or the extracted
deconvolved spectrum, as the input to various search engines.

Figure 181 shows an isotopically unresolved mass spectrum. The mass-spectral peaks
represent different charge states.
Figure 181. Isotopically unresolved mass spectrum of myoglobin

Figure 182 shows an isotopically resolved mass spectrum for one charge state.
Figure 182. Isotopically resolved mass spectrum of myoglobin for one charge state showing two components

Applying the Xtract Algorithm to an Isotopically Resolved Spectrum

Y To apply the Xtract algorithm to an isotopically resolved spectrum

1. Review the default settings for the Xtract algorithm on the Xtract Options page of the
Default Options Configuration dialog box as follows:
a. In the Workspace Options toolbar, click Default Options.

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Page for a Selected Spectrum

b. In the navigation pane, click Xtract Options.

c. When needed, modify the settings and click Save.
2. Open a raw data file.
3. Select a mass spectrum.
4. Click the Workspace Processing tab to open the Workspace Processing toolbar.
5. Click Xtract Deconvolution.
The application adds the Xtract page to the Info Bar.
6. Open the Xtract page.
The M/Z Range boxes are populated with the m/z range of the selected spectrum.
7. When necessary, modify the m/z range by entering a range within the selected spectrum.
8. Review and modify the settings in the Deconvolution Parameters area as appropriate.
9. Click Apply.
The application runs the Xtract algorithm.

Xtract Page for a Selected Spectrum

Use the Xtract page in the Info Bar to specify the parameter settings for the Xtract algorithm.

Y To display the Xtract page

1. Click the Workspace Processing tab to open the Workspace Processing toolbar.
2. Click Xtract Deconvolution.
Figure 183 shows the default settings on the Xtract page. The m/z range depends on the
selected spectrum.

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Xtract Page for a Selected Spectrum

Figure 183. Default settings on the Xtract page

Note The default parameters on the Xtract page are for protein and peptide
deconvolution in the positive polarity mode.

IMPORTANT Protein deconvolution in the FreeStyle application is limited to proteins of

less than 30 000 Da. To deconvolve proteins larger than 30 000 Da, use the BioPharma
Finder 4.1 application.

Table 58 describes the parameters for the Xtract page.

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Page for a Selected Spectrum

Table 58. Xtract page parameters for a spectrum (Sheet 1 of 4)

Parameter Description
Data Selection
M/Z Range Specifies the portion of the input spectrum that the Xtract
algorithm processes. The options are as follows:
• Min: Specifies the lowest end of the input spectrum
• Max: Specifies the highest end of the input spectrum
For example, if the total mass range of the spectrum is mass
100–2000, a setting of 300–500 for the m/z Range parameter
means that the Xtract algorithm processes only peaks with
masses between m/z 300 and 500.

Range: minimum m/z of the input spectrum to the maximum m/z

of the input spectrum

Default: minimum m/z of the input spectrum to the maximum

m/z of the output spectrum
Deconvolution Parameters
Output Mass Determines whether the Xtract algorithm returns a single peak at
either the monoisotopic mass or the monoisotopic MH+ mass for
each of the detected components. The options are as follows:
• M: Specifies that the results file contains a single peak for the
monoisotopic mass for each of the detected components. This
option generates masses without adducts.
• MH+: Specifies that the results file contains a monoisotopic
MH+ mass for each of the detected components. This option
generates masses with adducts.

Default: M

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Xtract Page for a Selected Spectrum

Table 58. Xtract page parameters for a spectrum (Sheet 2 of 4)

Parameter Description
Adduct Element Specifies the adduct ions used during ESI processing. Adduct ions
bring the charge to the molecule, and this charge converts it to an
ion. The options are as follows:
• H+ (1.00727663): Specifies that the adduct is hydrogen.
• K+ (38.9631585): Specifies that the adduct is potassium.
• Na+ (22.9892213): Specifies that the adduct is sodium.
• Custom: Specifies that the adduct is a charge carrier other
than hydrogen, potassium, or sodium. When you select this
option, a box opens so that you can type the mass of the
custom charge carrier.

Default: H+
Charge Range Specifies the lowest and highest charge state to be deconvolved.
The options are as follows:
• Low: Specifies the lowest charge state.
• High: Specifies the highest charge state.

For example, if you set this parameter range to 1–5, the Xtract
algorithm considers only charge states 1 through 5 for
deconvolution. It ignores charge states 6 and higher.

Default range: 5–50

Analyzer Type Specifies the type of mass analyzer that was used to obtain the
mass spectral data. Different types of mass analyzers have different
mass accuracies. The options are as follows:
• FT: Specifies a Fourier transform - ion cyclotron resonance
(FT-ICR) mass analyzer
• OT: Specifies an Orbitrap™ mass analyzer
• Sector: Specifies a magnetic-sector mass analyzer
• Quad: Specifies a quadrupole mass analyzer

Default: OT

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Xtract Page for a Selected Spectrum

Table 58. Xtract page parameters for a spectrum (Sheet 3 of 4)

Parameter Description
Rel. Abund. Threshold Specifies a threshold (as a percentage) below which the Xtract
(%) algorithm filters out data for data reporting.

This option sets a relative threshold as a percentage of the most

abundant component in the spectrum. The most abundant peak
in the deconvolved spectrum has a relative abundance of 100
percent, and all other peaks are calculated relative to that one.

In the Xtract Results table of the Xtract Results view, the

application shows only those components that are greater than or
equal to this relative abundance threshold in the deconvolved
spectrum. For example, if the highest peak has an absolute
abundance of 1000, the relative abundance is 1 percent, and no
peaks below an absolute abundance of 10 appear in the
deconvolved spectrum.

Zero (0) displays all results, and 100 displays only the most
abundant component.

Range: 0–100; default: 0

Isotope Table Specifies the type of isotope table to use.

Isotope tables simulate the distribution of isotopic peaks, in m/z,

for different choices of the monoisotopic mass. The Xtract
algorithm chooses the monoisotopic mass with the best fit
between the theoretical and the observed isotope distribution.

To generate an isotope table, the Xtract algorithm uses a chemical

formula to describe the type of molecule. You can choose one of
the following formulas:
• Protein: Uses an averagine formula typical for peptides and
proteins to generate the isotope table
• Nucleotide: Uses an elemental formula typical for nucleotides
to generate the isotope table

Default: Protein

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Xtract Page for a Selected Spectrum

Table 58. Xtract page parameters for a spectrum (Sheet 4 of 4)

Parameter Description
Negative Charge Indicates whether the data was acquired in positive charge mode
or negative charge mode during the ESI process.

You might want to use this option when you process compounds
that contain nucleotides like those found in DNA and RNA.
When you acquire these compounds in negative mode, the
resulting mass spectra are often clearer. Deprotonation of
nucleotides, which are acidic, occurs when the compound is
dissolved in a basic solution and negative voltage is applied to
produce negatively charged ions. The options are as follows:
• Selected: The data was acquired in negative charge mode.
• Cleared: The data was acquired in positive charge mode.

Default: Cleared
IMPORTANT Do not select Negative Charge if your data was
acquired in positive mode. The results will not be usable.
Min Num Detected Specifies the minimum number of charge states required to
Charge produce a component. No components with less than this
minimum number appear in the deconvolved spectrum.

Valid values: an integer greater than or equal to 1

Default: 3
Button
Load Defaults Resets all the parameters on the Xtract page to the default settings.
Apply Runs the Xtract algorithm on the selected spectrum.

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Deconvolved Spectrum View

Deconvolved Spectrum View

The Xtract algorithm transforms the source mass spectrum into a deconvolved mass spectrum
and displays it in the Deconvolved Spectrum view, labeled with mass units rather than with
the mass-to-charge ratio on the x axis. The spectrum that the application displays in the
Deconvolved Spectrum view is a mass spectrum that consists of the monoisotopic peaks of the
components that the Xtract algorithm detected. The range of the mass spectrum is from the
mass of the component with the lowest monoisotopic mass to the mass of the component
with the highest monoisotopic mass. Information about each component can be found in the
Xtract Results view.

Y To run the Xtract algorithm and display the Deconvolved Spectrum view

1. Select a mass spectrum for the Xtract algorithm to deconvolve.

2. Click the Workspace Processing tab to open the Workspace Processing toolbar.
3. Click Xtract Deconvolution.
The application adds the Xtract page to the Info Bar.
4. Open the Xtract page.
5. Set the deconvolution parameters on the Xtract page.
6. Click Apply.

Figure 184 shows the deconvolved spectrum for the myoglobin example.
Figure 184. Deconvolved Spectrum view showing the two monoisotopic peaks of myoglobin
(located at the endpoints)

The spectrum in the Spectrum View or MultiSpectrum View shows the multiple masses (see
Figure 187) used to create the deconvolved peak displayed in the Deconvolved Spectrum
view.

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Xtract Results View

Xtract Results View

The Xtract Results view displays the experimentally determined results for each monoisotopic
mass and charge state.

Xtract deconvolves isotopically resolved mass spectra—that is, spectra in which it is possible
to distinguish separate peaks for different isotopic compositions of the same component.
When the Xtract algorithm finishes processing, it displays the deconvolved spectrum in the
Deconvolved Spectrum View as a set of peaks in mass and relative intensity.

It also displays the component list in the Xtract Results view as a list of masses, intensities,
charge state information, and quality scores (Figure 185). The values in the columns represent
the outputs of the deconvolution. Each peak in the list is composed of isotopic clusters. Each
isotopic cluster in the original spectrum provides evidence for the peak in the deconvolved
spectrum.

Y To run the Xtract algorithm and display the Xtract Results view

1. Display the mass spectrum for the Xtract algorithm to deconvolve in the Spectrum or
MultiSpectrum view.
2. Click the Workspace Processing tab to open the Workspace Processing toolbar.
3. Click Xtract Deconvolution.
The application adds the Xtract page to the Info Bar.
4. Open the Xtract page, and set the parameters on the Xtract Page for a Selected Spectrum.
5. Click Apply.

Figure 185 shows the Xtract Results view for the two-component myoglobin example.
Figure 185. Xtract Results view in two parts (left and right) showing the 16 923 Da and 16 941 Da
components

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Results View

Table 59 describes the parameters for the Xtract Results view.

Table 59. Xtract Results view parameters (Sheet 1 of 2)
Parameter Description
No. An integer, starting at one, that labels the components in the
order of increasing monoisotopic mass.
Monoisotopic Mass The weighted average of the monoisotopic masses of each
charge state:

 ( Monoisotopic Mass of This Charge × Charge Normalized Intensity )

i
Monoisotopic Mass = -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Sum Intensity

where i is the sequential order of the charges in the Charge column of the expanded
Xtract table.
Sum Intensity The sum of the intensities of all the charge states and the
isotopes of a component.
Number of Charge States The number of charge states that the Xtract algorithm used
to calculate the monoisotopic mass.
Average Charge The average of the charges weighted by intensity.
Delta Mass The difference in mass (in daltons) of a component from the
mass of the most intense component.
Relative Abundance Displays the components that are above the relative
abundance threshold set by the Relative Abundance
Threshold parameter on the Xtract page. The application
assigns the largest peak in a deconvolved spectrum a relative
abundance of 100 percent. An abundance number in the
Relative Abundance column represents the intensity in the
same row of the Sum Intensity column divided by the
greatest intensity in the Sum Intensity column multiplied by
100.

For example, if the largest peak in a deconvolved spectrum

has an intensity of 1000, the application assigns it a relative
abundance of 100 percent. If the next most abundant peak
has an intensity of 500, the application assigns it an
abundance of 50 percent:
500
------------ × 100% = 50%
1000

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Xtract Results View

Table 59. Xtract Results view parameters (Sheet 2 of 2)

Parameter Description
Fractional Abundance The fractional abundance of a component, which is the
abundance for that peak relative to the total abundance of all
peaks in the spectrum, expressed as a percentage. The sum of
all fractional abundances of all peaks in a deconvolved
spectrum is 100 percent.
RT Range The retention time (in minutes), or the retention time range
(if the retention time was averaged), that produced the mass
spectrum that the Xtract algorithm deconvolved.

Click to expand the Xtract Results view for a component. The expanded list shows the
experimentally-determined parameters for the individual charge states of a component.

In the expanded list (see Figure 186), six new columns appear: Charge State, Calculated
Monoisotopic m/z, Monoisotopic Mass for This Charge, Mostabund m/z, Charge
Normalized Intensity, and Fit %. These values represent the isotopic clusters with different
charge states from the source spectrum that were used to produce the peak in the deconvolved
spectrum.

Figure 186 shows the Xtract Results view of Figure 185 with the 16 923 Da component
expanded to show the individual charge states.
Figure 186. Xtract Results view with the 16 923 Da component expanded to show results for the
nine individual charge states

Table 60 describes the parameters for the expanded Xtract Results view.
Table 60. Expanded Xtract Results view parameters (Sheet 1 of 2)
Parameter Description
Charge State The charge states of the individual isotopic clusters that
constitute the total number shown in the Number of Charge
States column.
Calculated Monoisotopic The mass-to-charge ratio of the monoisotopic mass that the
m/z Xtract algorithm calculated from the isotopic peak envelope
for a specific charge state.

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Results View

Table 60. Expanded Xtract Results view parameters (Sheet 2 of 2)

Parameter Description
Monoisotopic Mass For The mass (in daltons) that is equal to the calculated
This Charge monoisotopic m/z multiplied by the charge state, z.
Mostabund m/z The mass-to-charge ratio of the most abundant isotope, or
the height of the tallest peak in the isotopic distribution.
Charge Normalized The sum of the intensities of the isotopic peaks for a
Intensity particular charge state.
Fit % The quality of the match between a measured isotope pattern
and an averagine distribution of the same mass. This column
displays a value between 0 and 100 percent.
• 0 percent indicates a poor fit between the measured
pattern and the averagine pattern.
• 100 percent indicates that the observed peaks in the
measured isotope profile are absolutely identical to those
in a theoretical averagine distribution and that any
missing peaks fall below a restrictive threshold.

When you select any charge state row in the expanded Xtract Results view, the application
automatically highlights in blue the following items:
• The blue spectral line in the Spectrum View or MultiSpectrum View corresponding to
the most abundant m/z for the selected row, as shown in Figure 187 and Figure 188.
For example, in Figure 188, a blue spectral line marks the value of 770.6924
corresponding to the Mostabund m/z value of the selected row.
• The deconvolved peak in the Deconvolved Spectrum View.

You might have to right-click and choose Reset to Scale to see the blue spectral lines.

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Xtract Results View

Figure 187 shows the mass spectrum from Figure 181 with nine blue bars that indicate the
positions of the theoretical most abundant m/z for each of the nine charge states that the
Xtract algorithm used to deconvolve the spectrum.
Figure 187. Mass spectrum of myoglobin showing the nine charge states (blue bars) that the Xtract algorithm used to calculate
the 16923 Da monoisotopic mass

Figure 188 shows the theoretical most abundant m/z for the z=22 charge state of the
16923 Da component.
Figure 188. Mass spectrum at the z = 22 charge state showing the location (blue bar) of the theoretical most abundant m/z for
the 16 923 Da monoisotopic mass

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Applying the Xtract Algorithm to a Chromatogram

Applying the Xtract Algorithm to a Chromatogram

Y To apply the Xtract algorithm to a chromatogram

1. Review the default settings for the Xtract algorithm on the Xtract Options page of the
Default Options Configuration dialog box as follows:
a. In the Workspace Options toolbar, click Default Options.
b. In the navigation pane, click Xtract Options.
c. When needed, modify the settings and click Save.
2. Open a raw data file.
3. Select the Chromatogram view.
4. Click the Workspace Processing tab to open the Workspace Processing toolbar.
5. Click Xtract Deconvolution.
The application adds the Xtract page to the Info Bar.
6. Open the Xtract page.
The M/Z Range boxes are populated with the m/z range of the spectrum.
7. When necessary, modify the m/z range by typing the new start and stop values.
8. Review and modify the settings in the Xtract All Parameters area as appropriate.
9. Review and modify the settings in the Deconvolution Parameters area as appropriate.
10. Click Xtract All.
The application runs the Xtract algorithm.

It can take a few minutes depending on your filters and the amount of data in the
specified range.
When the deconvolution process completes, the application writes your file to the name
and location specified in the Xtract File Name box and records the extraction in the Audit
Trail.

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Xtract Page for a Selected Chromatogram

Xtract Page for a Selected Chromatogram

Use the Xtract page in the Info Bar to specify the parameter settings for the Xtract algorithm.

Y To display the Xtract page

1. Click the Workspace Processing tab to open the Workspace Processing toolbar.
2. Click Xtract Deconvolution.
Figure 189 shows the default settings on the Xtract page.
Figure 189. Default settings on the Xtract page

Note The default parameters on the Xtract page are for protein and peptide
deconvolution in the positive polarity mode.

IMPORTANT Protein deconvolution in the FreeStyle application is limited to proteins of

less than 30 000 Da. To deconvolve proteins larger than 30 000 Da, use the BioPharma
Finder 4.1 application.

Table 61 describes the parameters for the Xtract page.

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Page for a Selected Chromatogram

Table 61. Xtract page parameters for a chromatogram (Sheet 1 of 4)

Parameter Description
Data Selection
M/Z Range Specifies the portion of the input spectrum that the Xtract algorithm processes. The options are
as follows:
• Min: Specifies the lowest end of the input spectrum
• Max: Specifies the highest end of the input spectrum
For example, if the total mass range of the spectrum is mass 100–2000, a setting of 300–
500 for the m/z Range parameter means that the Xtract algorithm processes only peaks with
masses between m/z 300 and 500.

Range: minimum m/z of the input spectrum to the maximum m/z of the input spectrum

Default: minimum m/z of the input spectrum to the maximum m/z of the output spectrum
Xtract All Parameters
Xtract File Name Optional file name for the extracted file. When you do not specify a new file name, the
application uses the default file name.

Default: rawFileName_Xtract_dateTimeStamp
Example: Myoglobin_Xtract_2020-12-23_11-20-43-PM.raw
Scan Filter Deconvolves the mass spectra in the chromatogram for the selected scan filter. If no scan filter is
selected, the application extracts all of the scans.
Xtract What Specifies that the application create one result scan for each input scan for either of the
following:

Monoisotopic Masses: The resulting scan contains the singly charged (uncharged when MH+ is
not selected) monoisotopic masses that the application recognized in the input scan.

Full Pattern: The resulting scan contains the singly charged (uncharged when MH+ is not
selected) full pattern that the application recognized in the input scan.
Time Range Start and end time for the extraction.

Range: The minimum and maximum of the m/z range in the current spectra
Save Log Files Saves a log file for each input scan for which the Xtract deconvolution was performed.

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Xtract Page for a Selected Chromatogram

Table 61. Xtract page parameters for a chromatogram (Sheet 2 of 4)

Parameter Description
Deconvolution Parameters
Output Mass Determines whether the Xtract algorithm returns a single peak at either the monoisotopic mass
or the monoisotopic MH+ mass for each of the detected components. The options are as
follows:
• M: Specifies that the results file contains a single peak for the monoisotopic mass for each
of the detected components. This option generates masses without adducts.
• MH+: Specifies that the results file contains a monoisotopic MH+ mass for each of the
detected components. This option generates masses with adducts.

Default: M
Adduct Element Specifies the adduct ions used during ESI processing. Adduct ions bring the charge to the
molecule, and this charge converts it to an ion. The options are as follows:
• H+ (1.00727663): Specifies that the adduct is hydrogen.
• K+ (38.9631585): Specifies that the adduct is potassium.
• Na+ (22.9892213): Specifies that the adduct is sodium.
• Custom: Specifies that the adduct is a charge carrier other than hydrogen, potassium, or
sodium. When you select this option, a box opens so that you can type the mass of the
custom charge carrier.

Default: H+
Charge Range Specifies the lowest and highest charge state to be deconvolved. The options are as follows:
• Low: Specifies the lowest charge state.
• High: Specifies the highest charge state.

For example, if you set this parameter range to 1–5, the Xtract algorithm considers only charge
states 1 through 5 for deconvolution. It ignores charge states 6 and higher.

Default range: 5–50

Analyzer Type Specifies the type of mass analyzer that was used to obtain the mass spectral data. Different
types of mass analyzers have different mass accuracies. The options are as follows:
• FT: Specifies a Fourier transform - ion cyclotron resonance (FT-ICR) mass analyzer
• OT: Specifies an Orbitrap™ mass analyzer
• Sector: Specifies a magnetic-sector mass analyzer
• Quad: Specifies a quadrupole mass analyzer

Default: OT

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 13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Page for a Selected Chromatogram

Table 61. Xtract page parameters for a chromatogram (Sheet 3 of 4)

Parameter Description
Rel. Abund. Specifies a threshold (as a percentage) below which the Xtract algorithm filters out data for data
Threshold (%) reporting.

This option sets a relative threshold as a percentage of the most abundant component in the
spectrum. The most abundant peak in the deconvolved spectrum has a relative abundance of
100 percent, and all other peaks are calculated relative to that one.

In the Xtract Results table of the Xtract Results view, the application shows only those
components that are greater than or equal to this relative abundance threshold in the
deconvolved spectrum. For example, if the highest peak has an absolute abundance of 1000, the
relative abundance is 1 percent, and no peaks below an absolute abundance of 10 appear in the
deconvolved spectrum.

Zero (0) displays all results, and 100 displays only the most abundant component.

Range: 0–100; default: 0

Isotope Table Specifies the type of isotope table to use.

Isotope tables simulate the distribution of isotopic peaks, in m/z, for different choices of the
monoisotopic mass. The Xtract algorithm chooses the monoisotopic mass with the best fit
between the theoretical and the observed isotope distribution.

To generate an isotope table, the Xtract algorithm uses a chemical formula to describe the type
of molecule. You can choose one of the following formulas:
• Protein: Uses an averagine formula typical for peptides and proteins to generate the isotope
table
• Nucleotide: Uses an elemental formula typical for nucleotides to generate the isotope table

Default: Protein
Negative Charge Indicates whether the data was acquired in positive charge mode or negative charge mode
during the ESI process.

You might want to use this option when you process compounds that contain nucleotides like
those found in DNA and RNA. When you acquire these compounds in negative mode, the
resulting mass spectra are often clearer. Deprotonation of nucleotides, which are acidic, occurs
when the compound is dissolved in a basic solution and negative voltage is applied to produce
negatively charged ions. The options are as follows:
• Selected: The data was acquired in negative charge mode.
• Cleared: The data was acquired in positive charge mode.

Default: Cleared
IMPORTANT Do not select Negative Charge if your data was acquired in positive mode. The
results will not be usable.

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13 Running the Xtract Algorithm on Spectra and Chromatogram Data
Xtract Page for a Selected Chromatogram

Table 61. Xtract page parameters for a chromatogram (Sheet 4 of 4)

Parameter Description
Min Num Detected Specifies the minimum number of charge states required to produce a component. No
Charge components with less than this minimum number appear in the deconvolved spectrum.

Valid values: an integer greater than or equal to 1

Default: 3
Button
Load Defaults Resets all the parameters on the Xtract page to the default settings.
Xtract All Runs the Xtract algorithm on each input scan/spectrum specified by the Xtract All Parameters –
Time Range parameter.
Help Opens the Help topic for this page.

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 A

FreeStyle Default Settings

Use the pages of the Default Options Configuration dialog box to specify the initial default
settings that the FreeStyle application uses when you start the application.

Follow the procedures in these topics.

Contents
• Default Mass Precision Page
• Default Peak Detection Algorithm Page
• Default Number of Recently Used Files Page
• Default Raw Data Files Directory Page
• Default Elemental Composition Search Parameter Page
• Factory Default Template Page
• Default Library Search Parameters Page
• Workspace Options Page
• Xtract Options Page

Modifying, Saving, and Resetting the Default Configuration Options

Use the pages of the Default Options Configuration dialog box to modify some of the
parameter settings for the FreeStyle application.

Follow these procedures:

• To open the Default Options Configuration dialog box
• To display a specific page of the dialog box
• To open the Help topic for a specific page
• To save your new parameter settings

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Modifying, Saving, and Resetting the Default Configuration Options

• To cancel all your changes to the parameter settings

• To return all the configuration parameters to their factory default settings
• To apply the factory default layout to the active Workspace page

Y To open the Default Options Configuration dialog box

• In the Workspace Options Toolbar, click Default Options.

–or–
• Choose File > Default Options. See File Menu.
The dialog box opens to the Default Mass Precision page.

Y To display a specific page of the dialog box

In the navigation pane on the left, click the page link.

Y To open the Help topic for a specific page

Click Help at the bottom right of the page.

Y To save your new parameter settings

Click Save.
The Default Options dialog box closes, and the application saves all the modified
parameter settings.

Y To cancel all your changes to the parameter settings

Click Cancel.
The Default Options Configuration dialog box closes without changing the parameter
settings.

Y To return all the configuration parameters to their factory default settings

At the bottom left, click Revert All to Factory Default Values.

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 A FreeStyle Default Settings
Default Mass Precision Page

Figure 190. Location of the Revert All to Factory Default Values button
Restores the factory default
settings for the current page

Restores the factory Saves all of your settings

default settings for all and closes the dialog box
the dialog box pages
Cancels all of your custom
settings and closes the dialog box

Opens the Help topic for the

current page

Y To apply the factory default layout to the active Workspace page

1. In the navigation pane of the dialog box, click Factory Default Template.
2. At the top right of the Factory Default Template Page, click Revert to Factory Default
Values.

Default Mass Precision Page

Use the Default Mass Precision page to specify how many decimal places the application
displays, by default, and also the default mass tolerance.

Y To display the Default Mass Precision page

1. Open the Default Options Configuration dialog box (see Modifying, Saving, and
Resetting the Default Configuration Options).
2. In the navigation pane, click Mass Precision.

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Default Mass Precision Page

Figure 190 shows the Default Mass Precision page with its original default values, and
Table 62 lists the default Mass Precision page parameters.
Table 62. Default Mass Precision page parameters (Sheet 1 of 2)
Column Description
Mass Precision Specify the default number of decimal places in the mass-to-charge
ratios that the application displays in the Spectrum view and the
MultiSpectrum view.

Default:
• FTMS: 4
• ITMS: 2
• TQMS: 2
• SQMS: 1
• Sector: 4
• Unrecognized: 2

Range: 0–5
Mass Tolerance Specify the default mass tolerance.

The mass tolerance defines how close a measured mass must be to

the mass in the mass list to be considered the same mass.

Default:
• FTMS: 5.00 ppm
• ITMS: 0.50 amu
• TQMS: 0.50 amu
• SQMS: 1.00 amu
• Sector: 5.00 ppm
• Unrecognized: 0.50 amu

Range: 0.00–1000.00 ppm or 0.00–10.00 amu

Default Mass Specify the default mass tolerance units as follows:
Tolerance Units
• ppm: In parts per million. Select this option for FTMS and
sector mass analyzers because the peak width is proportional to
the mass-to-charge ratio. When you select ppm, the size of the
window around the reference mass is proportional to the
mass-to-charge ratio of the reference mass.
• amu: In units of the mass-to-charge ratio. When you select amu,
the size of the window around the reference mass remains the
same regardless of the mass-to-charge ratio of the reference mass.
• mmu: In units of one one-thousandth of an amu.

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 A FreeStyle Default Settings
Default Peak Detection Algorithm Page

Table 62. Default Mass Precision page parameters (Sheet 2 of 2)

Column Description
Button
Revert to Factory Returns the parameter settings on this page to their factory default
Default Values values.
Help Opens the Help topic for this page.

Default Peak Detection Algorithm Page

Use the Default Peak Detection Algorithm page to select the default peak detection algorithm
and its default parameters. For a description of these parameters, see these topics: Avalon Peak
Detection Page, Genesis Peak Detection Page, ICIS Peak Detection Page, or PPD Peak
Detection Page.

Y To display the Default Peak Detection Algorithm page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Peak Detection.

To save your custom settings, see Modifying, Saving, and Resetting the Default Configuration
Options.

Default Number of Recently Used Files Page

Use the Default Number of Recently Used Files page to set the number of recently used files
that the application displays in the Recent Items area of the File menu. By default, this
number is set to 10. You can change the setting to a value from 0 to 10.

Y To display the Default Number of Recently Used Files page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Recently Used Files.
The Default Number of Recently Used Files page appears (Figure 191).

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A FreeStyle Default Settings
Default Raw Data Files Directory Page

Figure 191. Default Number of Recently Used Files page

Table 63 describes the parameter and buttons on the Default Number of Recently Used Files
page. To save a custom setting, see Modifying, Saving, and Resetting the Default
Configuration Options.
Table 63. Default Number of Recently Used Files page parameter and buttons
Parameter Description
# of Recently Used Specifies the maximum number of files that the application displays
Files in the Recent Items area of the File menu.

Range: 0 to 10; default: 10

Button
Revert to Factory Returns the parameter settings on this page to their factory default
Default Values values.
Help Opens the Help to the topic for this page.

Default Raw Data Files Directory Page

Use the Default Raw Data Files Directory page to specify the default location where the
FreeStyle application looks for raw data files.

Y To display the Default Raw Data Files Directory page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Raw Files Directory (Figure 192).

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 A FreeStyle Default Settings
Default Elemental Composition Search Parameter Page

Figure 192. Default Raw Files Directory page

Table 64 describes the parameter and buttons on the Default Raw Files Directory page. To
save a custom setting, see Modifying, Saving, and Resetting the Default Configuration
Options.
Table 64. Default Raw Files Directory page parameter and buttons
Parameter Description
Select RawFiles Default: C:\Xcalibur\examples\data
Location
Button
Revert to Factory Returns the parameter settings on this page to their factory default
Default Values values.
Help Opens the Help to the topic for this page.

Default Elemental Composition Search Parameter Page

Use the Default Elemental Composition Search Parameter page to specify the default
elemental composition search parameters. For information about the basic parameter settings
and performing an elemental composition analysis, see Elemental Composition Page. To save
your custom settings, see Modifying, Saving, and Resetting the Default Configuration
Options. To use a custom periodic table for elemental composition analysis, see Using
Custom Periodic Table for Analysis.

Y To display the Default Elemental Composition Search Parameter page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Elemental Composition.

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A FreeStyle Default Settings
Factory Default Template Page

Figure 193 shows the Default Elemental Composition Search Parameter page. For
information about the parameter settings, see Elemental Composition Page.
Figure 193. Default Elemental Composition Search Parameter page

Factory Default Template Page

Use the Factory Default Template page to apply the factory default layout to the active
Workspace page (see Modifying, Saving, and Resetting the Default Configuration Options).
The settings on this page are read-only.

Y To display the Factory Default Template page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Factory Default Template (Figure 194).

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 A FreeStyle Default Settings
Factory Default Template Page

Figure 194. Factory Default Template page

Click to apply the factory default layout to

the active Workspace page.

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A FreeStyle Default Settings
Default Library Search Parameters Page

Default Library Search Parameters Page

Use the Default Library Search Parameters page to specify the default library search
parameters for the NIST libraries and the mzVault libraries. For a description of these
parameters, see Modifying a NIST Search from the NIST Search Page and Modifying an
mzVault Search from the mzVault Search Page. To save your custom settings, see Modifying,
Saving, and Resetting the Default Configuration Options.

Y To display the Default Library Search Parameters page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Library Search.
Figure 195 shows the factory default settings for the Default Library Search Parameters
page.
Figure 195. Default Library Search Parameters page

Y To display the NIST library parameters

On the Default Library Search page, select the NIST option in the Library Type area.

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 A FreeStyle Default Settings
Workspace Options Page

Y To display the mzVault library parameters

1. On the Default Library Search page, select the mzVault option in the Library Type area.
2. Make sure that the Library list in the Search List area includes the mzVault databases that
you want to search (see To specify the location of your local mzVault database files).

Workspace Options Page

Use the Workspace Options page to specify the following:
• The minimum trace height (in centimeters) for the Chromatogram View, Spectrum
View, or MultiSpectrum View
• The maximum number of scan filters to display in the Chromatogram view when
applying the auto filter command (see Adding Chromatogram Traces with the Auto Filter
Feature)

Note When you adjust the height of these views, if the height becomes smaller than
the set minimum value, a scrollbar automatically appears to the right of the views.

Y To display the Workspace Options page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Workspace Options.
Figure 196 shows the factory default settings for the Workspace Options page.

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A FreeStyle Default Settings
Workspace Options Page

Figure 196. Workspace Options page

Table 65 describes the parameters on the Workspace Options page of the Default Options
Configuration dialog box.
Table 65. Workspace Options page parameters (Sheet 1 of 3)
Parameter Description
Chromatogram and MultiSpectrum Windows
Minimum Trace Specifies the minimum height, in centimeters, for the traces in the
Height (cm) Chromatogram and MultiSpectrum views.

Default: 2.65; range: 0.5 to 15.0

Default Auto Filter
# Number of Auto Specifies the default number of filtered chromatograms that the
Filter application displays in the Chromatogram Ranges and
Chromatogram views.

Default: 8; range: 1 to 500

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 A FreeStyle Default Settings
Workspace Options Page

Table 65. Workspace Options page parameters (Sheet 2 of 3)

Parameter Description
Nearby Precursor

Use this feature to label the spectrum peaks with related data-dependent scans within the
specified time range of the current scan. A related data-dependent scan is an MSn+1 stage
data-dependent scan for a precursor ion that matches the m/z value of the current peak
within the specified mass tolerance. See Using the Nearby Precursors Marker.
Entire File When selected, sets the search to every scan in the file (no time range
limit).

Default: Clear
Backward RT Specifies the first time point for the search:
RTcurrent scan – Specified time in minutes

Default: 1.00 min; range: 0.00 to 10.00 min

Not available when the Entire File check box is selected.

Forward RT Specifies the last time point for the search:
RTcurrent scan – Specified time in minutes

Default: 1.00 min; range: 0.00 to 10.00 min

Not available when the Entire File check box is selected.

Maximum Allowed Specifies the maximum number of matching data-dependent scans
Number of Hits to display or average. If the application finds more than this
maximum number, it displays the data-dependent scans with the
nearest RT to the precursor “survey” scan.

Default: 5; range: 1 to 50
Nearby Precursor Specifies whether double-clicking a marker in the precursor “survey”
Plot Type scan opens an average spectrum of the matching data-dependent
scans in another Spectrum view or opens a MultiSpectrum view with
a separate plot for each matching data-dependent scan.

Options: Average (default) or MultiSpectrum

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Workspace Options Page

Table 65. Workspace Options page parameters (Sheet 3 of 3)

Parameter Description
Mass Tolerance Specifies how close the m/z value of the precursor ion for the
data-dependent scan must be to the m/z value of the current
spectrum peak.

Default: Use Default is selected—the application uses the mass

tolerance of the mass spectrometer.

Use Default check box is clear—the settable range is from 0.00 to

1000.00 (amu, mmu, or ppm).
Default Y Axis Options

Use this feature to set the Y-Scale units as Relative or Absolute for each detector type.
Change the Y Axis units of individual traces of the Chromatogram view by enabling the
Separate Axis icon in the Y Axis area in Display Options toolbar.
MS Data The default Y-Scale unit for MS Data is Relative.

Select the check box for MS Data to change the default setting to
Absolute scale.
PDA/UV Data By default, the Y-Scale unit for PDA and UV Data is set to Absolute.

Clear the default check box to view the PDA and UV data in
Relative scale.
Other Data By default, the Y-Scale unit for Other Data is set to Absolute.

Clear the default check box to view the Other data in Relative scale.
Startup Dialog Options
Don’t Show Again No longer displays this startup page when you launch the FreeStyle
application.

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 A FreeStyle Default Settings
Xtract Options Page

Xtract Options Page

Use the Xtract Options page to specify the default Xtract parameters. For more information,
see Chapter 13, “Running the Xtract Algorithm on Spectra and Chromatogram Data.”

Y To display the Xtract Options page

1. In the Workspace Options toolbar, click Default Options.

The Default Options Configuration dialog box opens.
2. In the navigation pane, click Xtract Options.

Figure 197 shows the Xtract Options page with its original default values.
Figure 197. Default Xtract Options page

Table 66 lists the default Xtract parameters.

Table 66. Default parameters for the Xtract algorithm (Sheet 1 of 3)
Parameter Description
Fit Factor (%) Measures the quality of the match between a measured
isotope pattern and an averagine distribution of the same
mass, as a percentage.
• 0% requires a low fit only.
• 100% means that the measured isotope profile is
identical to the theoretical averagine isotope distribution.

Range: 0–100; default: 80

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Table 66. Default parameters for the Xtract algorithm (Sheet 2 of 3)

Parameter Description
Remainder Threshold (%) Specifies the height of the smaller overlapping isotopic
cluster (as a percentage) with respect to the height of the
most abundant isotopic cluster when the Xtract algorithm
attempts to resolve overlapping isotopic clusters.

For example, if one isotopic cluster in a spectrum has an

abundance of 100 and you set the Remainder Threshold
parameter to 30 percent, the Xtract algorithm ignores any
overlapping clusters with an abundance less than 30.

Range: 0–100; default: 25

Consider Overlaps Determines whether the Xtract algorithm is more tolerant of
errors when the spectrum intensity is significantly higher
than expected for the theoretical isotopic cluster.
• Selected: The Xtract algorithm is more tolerant of errors
when the spectrum intensity is significantly higher than
expected for the theoretical isotopic cluster. Because this
option can lead to increased false positives, select it only
in cases where you expect overlapping isotopic clusters in
a data set.
• Cleared: The Xtract algorithm is less tolerant of errors
when the spectrum intensity is significantly higher than
expected for the theoretical isotopic cluster.

Default: Selected
Minimum Intensity Specifies a minimum intensity threshold to filter out possible
background noise, even when you set the S/N Threshold
parameter to zero.

Range: 0–9999; default: 1

Expected Intensity Error Specifies the permissible percentage of error allowed in
calculating the ratio of the most abundant isotope to the next
isotope that is higher in mass in the isotope series.

Range: 0–9999; default: 3

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 A FreeStyle Default Settings
Xtract Options Page

Table 66. Default parameters for the Xtract algorithm (Sheet 3 of 3)

Parameter Description
Resolution at 400 m/z Defines the resolution of the source spectrum at m/z 400.

This parameter is not needed if the Xtract algorithm

deconvolves FTMS, Orbitrap, or Exactive data because the
data contains the instrument information in the spectrum.
You must set this parameter for all other spectrum types and
for exported spectrum files (in -qb.raw data file format),
which lack instrument information.

Range: 6000–240 000; default: 60 000

S/N Threshold Specifies a signal-to-noise (S/N) threshold, x, above which
the Xtract algorithm considers a measured peak to be a real
(accepted) peak. The Xtract algorithm ignores peaks below
this threshold.

Any spectral peak must be x times the intensity of the

calculated noise for that spectrum before the Xtract
algorithm considers it.

Range: 0–9999; default: 3

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 B

Scan Filters and Scan Headers

The scan method used for each scan is recorded as a scan event. The data system creates scan
filters from the scan-event settings. You can select or create scan filters to apply processing to a
subset of the scans in the raw data file. Scan headers provide important information about the
scan.

Contents
• Scan Filter Parameters
• Scan Headers and Scan Header Abbreviations

Scan Filter Parameters

Table 67 lists the scan filter parameters. Use only the fields that apply to your mass
spectrometer. You can define additional scan filters by adhering to the following scan filter
format.

Note Not all features are applicable for every mass spectrometer.

Table 67. Scan filter format (Sheet 1 of 4)

Feature Option Interpretation
Polarity +, – Positive or negative.
Data type p, c Profile or centroid.
Dependent scans d, !d Include dependent scans or exclude dependent scans.
TurboScans t, !t Include TurboScan scans or exclude TurboScan scans.
Source CID cid, !cid Include Source CID scans or exclude Source CID scans.

(Source CID scans are scans for ions that are produced by
collision-induced dissociation in the ion source.)
Scan type FULL, Z, SIM, SRM, Full scan, ZoomScan, selected ion monitoring (SIM), selected
CRM, Q1MS, Q3MS reaction monitoring (SRM), consecutive reaction monitoring
(CRM), Q1 quadrupole analysis, or Q3 quadrupole analysis.

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Scan Filter Parameters

Table 67. Scan filter format (Sheet 2 of 4)

Feature Option Interpretation
Scan mode ms, ms2, ms3, ... MS10 MSn where n = 1 to 10

Each order can be followed by the appropriate number of

parents. The parents can also be omitted.

Example: “ms3 345.3, 253.2” indicates an MS3 scan with

parents with m/z 345.3 and 253.2.
pr Parent (followed by the product mass)
cng Constant neutral gain (followed by the mass of the neutral)
cnl Constant neutral loss (followed by the mass of the neutral)
Mass Analyzer ITMS, TQMS, SQMS, Ion trap, triple-quadrupole, single-quadrupole, time-of-flight,
TOFMS, FTMS, Sector Fourier transform, or magnetic sector mass spectrometry.
Photo Ionization pi, !pi Include photo ionization scans or exclude photo ionization
scans.
Compensation Voltage cv, !cv Include compensation voltage scans or exclude compensation
voltage scans.
Detector Valid det, !det Include detector valid scans or exclude detector valid scans.
Enhanced E, !E Include enhanced scans or exclude enhanced scans.
Wideband w, !w Include wideband scans or exclude wideband scans.
Supplemental Activation sa, !sa Include supplemental activation scans or exclude supplemental
activation scans.
Multistate Activation msa, !msa Include multistate activation scans or exclude multistate
activation scans.
Product masses or mass [m1a–m1b, m2a–m2b, Scans with a specific mass range or mass ranges, such as SIM,
range of scan m3a–m3b, ...] SRM, and CRM.

Example: [50.00–1500.00] for a scan from m/z 50.00 to

1500.00

If a scan is exactly 1 Da wide, it appears as a single value (the

center mass). This is typical for SIM, SRM, and CRM. Filters
for precursor ions in dependent scans are matched with a
tolerance of m/z 1.0 so that minor differences in precursor mass
measurements from scan to scan do not give different filters.
Segment/scan event {segment, scan number} Example: “{3, 4} + c ms” indicates segment 3, scan event 4 for a
number pairs positive centroid MS scan

The braces { } are required.

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 B Scan Filters and Scan Headers
Scan Filter Parameters

Table 67. Scan filter format (Sheet 3 of 4)

Feature Option Interpretation
Ionization mode APCI, ESI, EI, CI, NSI, Atmospheric pressure chemical ionization (APCI), electrospray
FAB, TSP, FD, MALDI, (ESI), electron ionization (EI), chemical ionization (CI),
GD, PSI nanoelectrospray ionization (NSI), fast atom bombardment
(FAB), thermospray ionization (TSP), field desorption (FD),
matrix-assisted laser desorption ionization (MALDI), or glow
discharge (GD), Paper Spray Ionization (PSI).

Example: “+ c ESI ms” indicates a positive centroid electrospray

MS scan.
Corona on/off corona, !corona Corona on or corona off.

Example: “+ APCI !corona ms” indicates a positive centroid

APCI scan with the corona off.
Detector value “det=## .##” Detector value is ## .## with no spaces.

Example: “+ ESI det= –800.0” indicates a positive electrospray

scan at –800.0 detector units (usually volts).
MS/MS and MSn CID mass@energy Mass is the precursor mass and energy is the CID relative
energies energy (no units).

Example: “– c ms2 [email protected]” indicates a negative centroid

MS/MS scan of m/z 196.1 at 25.0 units of CID energy.
Quadrupole Q1MS, Q3MS Q1 quadrupole or Q3 quadrupole.
identification
Example: “+ c ESI Q3MS” indicates a positive centroid
electrospray MS scan using Q3 quadrupole.
Accurate mass AM, !AM, AMI, AME Include accurate mass scans, exclude accurate mass scans,
include accurate mass internal, or include accurate mass
external.
Ultra u, !u Include ultra scans or exclude ultra scans.
Sector BSCAN, !BSCAN Include magnetic sector scans or exclude magnetic sector scans.
ESCAN, !ESCAN Include electric sector scans or exclude electric sector scans.
LOCK lock, !lock Include lock scans or exclude lock scans.
Multiplex msx, !msx Include multiplexing scans or exclude multiplexing scans.
Electron capture ecd, !ecd Include electron capture dissociation or exclude electron
dissociation capture dissociation.
Multi-photon mpd, !mpd Include photo dissociation or exclude photo dissociation.
dissociation

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B Scan Filters and Scan Headers
Scan Headers and Scan Header Abbreviations

Table 67. Scan filter format (Sheet 4 of 4)

Feature Option Interpretation
Pulsed dissociation pqd, !pqd Include pulsed dissociation scans or exclude pulsed dissociation
scans.
Electron transfer etd, !etd Include electron transfer dissociation scans or exclude electron
dissociation transfer dissociation scans.
NPTR nptr, !nptr Include NPTR, Exclude NPTR
Higher-energy CID hcd, !hcd Include higher energy CID scans or exclude higher energy CID
scans.
Source SID sid, !sid Include source SID scans or exclude Source SID scans.

(Source SID scans are surface-induced scans.)

Isolation width iw ##, ## Isolation width value at ##, ##

Scan Headers and Scan Header Abbreviations

Use the Scan Header dialog box to select what parameters appear in the scan header of the
Spectrum View or MultiSpectrum View.

Y To display the Scan Header dialog box

1. Click the Spectrum View or MultiSpectrum View to select it.

2. Click the Display Options tab to open the Display Options toolbar.
3. Click Scan Header.

Figure 198 shows the default scan header selections.

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 B Scan Filters and Scan Headers
Scan Headers and Scan Header Abbreviations

Figure 198. Default scan header selections

Table 68 lists the parameters that you can display in the scan header. The parameters
displayed in the Scan Header dialog box depend on the data acquisition settings for the mass
spectrometer used to acquire the raw data file.
Table 68. Scan header parameters and their abbreviations (Sheet 1 of 2)
Parameter Abbreviation before the value
Short filename NA
Scan Number #
Retention time RT
Average Number of Scans AV
Normalize Intensity NL
Scan Filter String T:
Polarity NA

(P: when the Scan Filter String is not

selected)
Background Subtraction Scan Numbers SB
Mass Ranges [...]

(MR: [...] when the Scan Filter String is

not selected)
Total Ion Current TIC:
Scan Low Mass SLM:

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Scan Headers and Scan Header Abbreviations

Table 68. Scan header parameters and their abbreviations (Sheet 2 of 2)

Parameter Abbreviation before the value
Scan High Mass SHM:
Scan Start Time (Min) SST (Min):
Base Peak Intensity BPI:
Base Peak Mass BPM:
Multiple Injection Multiple Injection::
Multi Inject Info Multi Inject Info::
AGC (automatic gain control—On, Off, or AGC::
predicted)
Micro Scan Count MSC:
Ion Injection Time (ms) IIT
Scan segment SS:
Scan Event SE:
Master Index Master Index::
Charge State CS:
Reagent Ion AGC Full parameter name
Reagent Ion Injection Time (msec) Full parameter name
Elapsed Scan Time (sec) EST
API Source CID Energy Full parameter name
Average Scan By Instrument ABSI
Charge State CS
Monoisotopic Ion M/Z Full parameter name
MSn Isolation Width (n = 2 to 10) (m/z) Full parameter name
FT analyzer settings Full parameter names
FT analyzer message Text message
FT resolution Full parameter names
Conversion parameters Full parameter names

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 C

One- and Three-Letter Abbreviations for Amino Acid

Residues
Table 69 lists common one- and three-letter abbreviations for amino acid residues that you
enter in the peptide/protein Formula box on the Isotope Simulation Page.
Table 69. Common one- and three-letter abbreviations for amino acid residues (Sheet 1 of 2)
One letter Name Formula Three letter
A Alanine C3H5NO Ala
C Cysteine C3H5NOS Cys
D Aspartate C4H5NO3 Asp
E Glutamate C5H7NO3 Glu
F Phenylalanine C9H9NO Phe
G Glycine C2H3NO Gly
H Histidine C6H7N3O His
I Isoleucine C6H11NO Ile
K Lysine C6H12N2O Lys
L Leucine C6H11NO Leu
M Methionine C5H9NOS Met
N Asparagine C4H6N2O2 Asn
O Ornithine C5H11N2O Orn
P Proline C5H7NO Pro
Q Glutamine C5H8N2O2 Gln
R Arginine C6H12N4O Arg
S Serine C3H5NO2 Ser

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C One- and Three-Letter Abbreviations for Amino Acid Residues

Table 69. Common one- and three-letter abbreviations for amino acid residues (Sheet 2 of 2)
One letter Name Formula Three letter
T Threonine C4H7NO2 Thr
V Valine C5H9NO Val
W Tryptophan C11H10N2O Trp
Y Tyrosine C9H9NO2 Tyr

Table 70 lists less common three-letter abbreviations for amino acid residues to enter in the
peptide/protein formula for the simulated spectrum.
Table 70. Less common three-letter abbreviations for amino acid residues (Sheet 1 of 3)
Three letter Name Formula
Abu 2-Aminobutyric acid C4H7NO
(2-aminobutanoic acid)
Aec Aminoethylcysteine C5H10N2OS
Aib Aminoisobutyric acid C4H7NO
Aln -- C13H11NO
Aly Alveolysin C12H22N2O6
Amc -- C6H10N2O2S
Bcy -- C10H11NOS
Bgl -- C12H13NO3
Bly -- C16H26N4O3S
Bse -- C10H11NO2
Bth -- C11H13NO2
Cmc Carboxymethylcysteine C5H7NO3S
Cml -- C8H14N2OS
Cph Chlorophenylalanine C9H8NOCl
Cya Cysteic acid C3H5NO4S
Dha Dehydroalanine C3H3NO
Dhb Dehydro-2-aminobutyric acid C4H5NO
Dpr D-proline C5H5NO
Dty Diiodotyrosine C9H7NO2I2
Fcy -- C18N29NOS
Fph -- C9H8NOF
Ftr -- C12H10N2O2

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 C One- and Three-Letter Abbreviations for Amino Acid Residues

Table 70. Less common three-letter abbreviations for amino acid residues (Sheet 2 of 3)
Three letter Name Formula
Gaa -- C4H7NO
Gcg -- C5H5NO4
Gla Carboxyglutamic acid C6H7NO5
Glp -- C5H5NO2
Hse Homoserine C4H7NO2
Hsl Homoserine lactone C4H5NO
Hya Beta-hydroxyaspartate C4H5NO4
Hyg Hydroxyglycine C5H7NO4
Hyl Hydroxylysine C6H12N2O2
Hyp Hydroxyproline C5H7NO2
Ils Isolysine C9H18N2O
Ity Iodotyrosine C9H8NO2I
Iva Isovaline C5H9NO
Mar -- C7H14N4O
Mas -- C5H7NO3
Mbt -- C17H17NO2
Mes -- C5H9NO3S
Mga -- C6H10N2O2
Mgl -- C6H9NO3
Mhi -- C7H9N3O
Mls -- C7H14NO
Mme -- C6H11NOS
Mph -- C10H11NO
Mso Methioninesulfoxide C5H9NO2S
Mty C10H11NO2
Nle Norleucine C6H11NO
Nls Norlysine C12H15N3O2
Pal -- C8H8N2O
Pcy -- C19H35NO2S
Pec -- C10H12N2OS
Pip 2-Piperidinecarboxylic acid C6H9NO

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C One- and Three-Letter Abbreviations for Amino Acid Residues

Table 70. Less common three-letter abbreviations for amino acid residues (Sheet 3 of 3)
Three letter Name Formula
Psr Phosphoserine C3H6NO5P
Pth Phosphothreonine C4H8NO5P
Pty Phosphotyrosine C9H10NO5P
Pyr Pyroglutamic acid C5H5NO2
Sar Sarcosine C3H5NO
Sas -- C8H8NO5
Tml E-amino trimethyl-lysine C9H19N
Tys Tyrosinesulfonic acid Tyr (SO3H) C9H9NO5S

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 D

Common Isotopes
Table 71 lists the exact mass and natural abundance for some common isotopes.
Table 71. Common elements with multiple stable isotopes
Element Isotope symbol Mass (Da)a %Abundanceb
79
Bromine Br 78.918336 50.69
81
Br 80.916290 49.31
12C
Carbon 12.000000 98.93
13C 13.003354 1.07
40
Calcium Ca 39.962591 96.95
42Ca 41.958622 0.65
44Ca 43.955485 2.086
35
Chlorine Cl 34.968853 75.77
37Cl 36.965903 24.23
Potassium 39K 38.963708 93.20
41
K 40.961825 6.73
14N
Nitrogen 14.003074 99.63
15N 15.00011 0.37
16
Oxygen O 15.994915 99.76
18O 17.999159 0.20
Sulfur 32S 31.972071 95.02
33
S 32.971459 0.75
34S 33.967867 4.21
a
Mass values are for reference only.
b
Isotopes with a natural abundance below 0.20% are not listed. Percent abundance values are for reference only.

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