MALDI

MATRIX ASSISTED LASER DESORPTION
IONIZATION TECHNIQUE
GUIDED BY: PRESENTED BY:
Prof.Dr.Shantha Kumari R.Keerthi (1st
M.Pharm)
M.Pharm,Ph.D Dept: Pharmaceutical Analysis.

CONTENTS:
 Introduction
 Matrix
 Laser
 Sample preparation
 Mechanism of MALDI
 Mass spectrometer
 Reproducibility and Performance
 Uses of MALDI

INTRODUCTION:
 Matrix-assisted laser desorption/ionization
 Soft ionization technique used in mass
spectrometry
 Analysis of bio molecules and large organic
molecules
 The ionization is triggered by a laser beam

MALDI is a two step process:
 First, Desorption is triggered by a UV laser
beam. Matrix material heavily absorbs UV
laser light leading to the ablation of the upper
layer of the matrix material. Hot plume gets
produced during ablation.
 Second, the analyte molecules are ionized in
the hot plume. Ablated species may
participate in the ionization of analyte .

Matrix:
Matrix consists of crystallised molecules of
which the three most commonly used are
 3,5 dimethoxy -4-hydroxy cinnamic acid
(sinapinic acid)
 α-cyano-4-hydroxycinnamic acid
(alpha-cyano or alpha-matrix)
 2,5 dihydroxy benzoic acid (DHB )

Considerations:
 They are of a fairly low molecular weight (to allow easy
vaporization), but are large enough (with a low enough
vapor pressure) not to evaporate during sample
preparation or while standing in the spectrometer.
 They are often acidic, therefore act as a proton source
to encourage ionization of the analyte.
 They have a strong optical absorption in either the UV
or IR range, so that they rapidly and efficiently absorb
the laser irradiation.
 They are functionalized with polar groups, allowing
their use in aqueous solutions.
 They typically contain a chromophore.

 The Matrix solution is mixed with the analyte
Eg: Protein sample.
 A mixture of water and organic solvent allows
both hydrophobic and water soluble
molecules to dissolve into the solution.
 This solution is spotted into a MALDI plate.
 The solvents vaporized , leaving only re-
crystallised matrix, but now with analyte
molecules embedded into MALDI crystals.
 The matrix and analyte are said to be co-
crystallised.
 Co-crystallization is a key issue in selecting a
proper matrix

Laser:
 MALDI techniques typically employ the use of UV
lasers such as nitrogen lasers (337 nm) and
frequency-tripled and quadrupled Nd:YAG lasers
(355 nm and 266 nm respectively).
 Although not as common, infrared lasers are used
due to their softer mode of ionization.
 IR-MALDI also has the advantage of greater material
removal (useful for biological samples), less low-
mass interferences, and compatibility with other
matrix-free laser desorption mass spectrometry
methods.

MALDI Sample Preparation
S.NO
Matrix Application
1 2,5-Dihydroxybenzoic acid (DHB) Peptides, proteins, lipids, and
oligosaccharides
2 3,5-Dimethoxy-4-hydroxycinnamic
acid (sinapinic acid)
Peptides, proteins, and
glycoprotein
3 a-Cyano-4-hydroxycinnamic acid
(CHCA)
Peptides, proteins, lipids, and
oligonucleotides

The mechanism of MALDI
 Done in three steps..
 (i) Formation of a Solid Solution
 (ii) Matrix Excitation
 (iii) Analyte Ionization

(i) Formation of a 'Solid Solution':
 It is essential for the
matrix to be in access thus leading to the
analyte molecules being completely isolated
from each other.
 This eases the formation of the homogenous
'solid solution' required to produce a stable
desorption of the analyte.

 (ii) Matrix Excitation:
 The laser beam is focussed onto the surface
of the matrix-analyte solid solution.
 The chromophore of the matrix couples with
the laser frequency causing rapid vibrational
excitation, bringing about localised
disintegration of the solid solution.
 The clusters ejected from the surface consists
of analyte molecules surrounded by matrix
and salt ions.
 The matrix molecules evaporate away from
the clusters to leave the free analyte in the
gas-phase.

 (iii) Analyte Ionisation:
 The photo-excited matrix molecules are
stabilised through proton transfer to the
analyte.
 Cation attachment to the analyte is also
encouraged during this process.
 It is in this way that the characteristic [M+X]+
(X= H, Na, K etc.) analyte ions are formed.
 These ionisation reactions take place in the
desorbed matrix-analyte cloud just above the
surface.
 The ions are then extracted into the mass
spectroscopy for analysis

Mass spectrometer
• Sample target for a MALDI mass spectrometer
• The type of a mass spectrometer most widely used with
MALDI is the TOF (time-of-flight mass spectrometer),
mainly due to its large mass range.
• The TOF measurement procedure is also ideally suited to
the MALDI ionization process since the pulsed laser takes
individual 'shots' rather than working in continuous
operation.
• MALDI-TOF instruments are typically equipped with an "ion
mirror", deflecting ions with an electric field, thereby
doubling the ion flight path and increasing the resolution.

 Today commercial reflectron TOF
instruments reach a resolving power m/Δm of
well above 20,000 FWHM ( full-width half-
maximum , Δm is defined as the peak width
at 50% of peak height. )
 MALDI-FT-ICR MS has been demonstrated to
be a useful technique where high resolution
MALDI-MS measurements are desired.

Reproducibility and
performance:
 The sample preparation for MALDI is
important for both sensitivity, reproducibility
and quantification of mass analysis.
 Inorganic salts which are also part of protein
extracts interfere with the ionization process.
 The salts can be removed by solid phase
extraction or by washing the dried droplet
MALDI spots with cold water.
 Both methods can also remove other
substances from the sample.

Uses of MALDI :-
 Used to characterize and identify large
molecules
 Used in pharmaceutical for QC, monitoring of
enzyme reactions
 Used in DNA sequencing for forensics
 Used to identify different strains of viruses to
help develop vaccines

What’s its future?
 Will help revolutionize the medical world and
will help lead to treatments for many diseases
 Will be useful for DNA sequencing, thus can
be useful for forensic investigations

Sources
•A text book of Organic spectroscopy by William Kemp.
•http://www.psrc.usm.edu/mauritz/maldi.html
•http://www.psrc.usm.edu/macrog/maldi.htm
•http://www.metabion.com/techinfo/z-maltof.html
•http://www.lsc.psu.edu/stf/imsc/MaldiTof.html
•http://www.laserscience.com/maldi_tof.htm

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