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Maldi (matrix attested laser desorption ionization technique
The document presents an overview of the matrix-assisted laser desorption/ionization (MALDI) mass spectrometry technique, focusing on its mechanism, preparation, and applications. MALDI is primarily used for analyzing biomolecules and large organic molecules by utilizing a matrix material that absorbs laser light to facilitate ionization. Its effectiveness in microbial identification and potential future applications in DNA sequencing highlight its significance in fields such as pharmaceuticals and forensics.
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Maldi (matrix attested laser desorption ionization technique
- 1. Presented By Mr. PritamP. Kolge FirstYear M. Pharm Department Of Pharmaceutical Quality Assurance BharatiVidyapeeth College Of Pharmacy, Kolhapur MATRIX ATTESTED LASER DESORPTION IONIZATION TECHNIQUE 1 Guided By Mr. D.V. Mahuli Assistant Professor Department Of Pharmacology
- 2. Contents 2 Introduction Matrix Laser Sample preparation Mechanism of MALDI Mass spectrometer Reproducibility and Performance Uses of MALDI
- 3. Introduction 3 Matrix-assisted laserdesorption/ionization Soft ionization technique used in mass spectrometry Analysis of bio molecules and large organic molecules The ionization is triggered by a laser beam It is used to determine the molecular weight of Peptides, Proteins,Antibodies upto size to 300 kDa
- 4. MALDI is twostep process 4 First, Desorption is triggered by a UV laser beam. Matrix material heavily absorbs UV laser light leading to the ablation of the upperlayer of the matrix material. Hot fumes gets produced during ablation. Second, the analyte molecules are ionized in the hot fumes.Ablated species may participate in the ionization of analyte.
- 5. Matrix 5 Matrix consists ofcrystallised molecules of which the most commonly used are 3,5 dimethoxy -4-hydroxy cinnamic acid(Sinapinic acid) 2,5 dihydroxy benzoic acid (DHB) Nicotinic acid Urea Cinnamic acid derivatives
- 6. Considerations of Matrixmaterial 6 They are of a fairly low molecular weight (to allow easy, vaporization/Ablation) 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 solution
- 7. 7 They typicallycontain a chromophore The Matrix solution is mixed with the analyte Eg: Protein sample. Solution is spotted into a MALDI plate. The matrix and analyte are said to be co-crystallised. Co-crystallization is a key issue in selecting a proper matrix
- 8. Laser 8 MALDI techniquestypically employ the Use of UV lasers such as nitrogen lasers (337 nm) and frequency- tripled and quadrupled 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.
- 9. MALDI Sample Preparation 9 Sr.No. Matrix Application 1 2,5 dihydroxy benzoic acid (DHB) Peptides, Proteins, Lipids, and Oligosaccharides 2 3,5 dimethoxy – 4 hydroxy cinnamic acid (Sinapinic acid) Peptides, Proteins, and Glycoprotein 3 a-cyano-4-hydroxycinnamic acid (CHCA) Peptides, Proteins, Lipids, Oligonucleotides
- 10. Mechanism of MALDI 10 Themechanism of MALDI Done in three steps… 1. Formation of a Solid Solution 2. Matrix Excitation 3. Analyte lonization
- 11. 1. Formation ofsolid solution 11 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.
- 12. 2. Matrix Excitation 12 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.
- 13. 3. Analyte Ionisation 13 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
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- 16. Mass Spectrometer 16 Sampletarget for a MALDI mass spectrometer The type of a mass spectrometer most widely used with MALDI is theTOF (time-of-flight mass spectrometer),mainly due to its large mass range. TheTOF 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 "ionmirror", deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution.
- 17. 17 Today commercialreflectronTOF instruments reach a resolving power m/Am of well above 20,000 FWHM (full-width half-maximum,Am is defined as the peak width at 5o% of peak height.) MALDI-FT-ICR (Ion Cyclotron Resonance) MS has been demonstrated to be a useful technique where high resolution MALDI-MS measurements are desired.
- 18. Reproducibility and Performance 18 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.
- 19. Uses of MALDI 19 Used to characterize and identify large molecules Used in pharmaceuticals , monitoring of enzyme reactions Used in DNA sequencing for forensics Used to identify different strains of viruses to help develop vaccines
- 20. What it’s Future 20 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
- 21. Conclusion 21 Matrix-assisted laserdesorption/ionization has become a gold standard for microbial identification in clinical microbiology laboratories. Matrix-assisted laser desorption/ionization has recently emerged as a rapid and accurate identification method for bacterial species. Effective tool for the rapid identification of arthropods, including tick vectors of human diseases.
- 22. References 22 A textbook of Organic spectroscopy byWlliam Kemp. http://www.psrc.usm.edu/mauritz/maldi.html http://www.psrc.usm.edu/macrog/maldi. htm Slideshare https://youtu.be/RuwbeA22rew
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