Maldi (matrix attested laser desorption ionization technique

MALDI...

This Presentation Contain following...
#Introduction
#Matrix and examples
#Considerations of Matrix Material
#MALDI Sample Preparation
#Mechanism of MALDI
#Mass Spectrometer
#Reproducibility and Performance
#Uses of MALDI
#Conclusion
#References
Thanks For Help and Guidance of Mr. D.V. Mahuli Sir and Mr. V.T. Pawar Sir

1. Presented By
Mr. Pritam P. Kolge
First Year M. Pharm
Department Of Pharmaceutical Quality
Assurance
Bharati Vidyapeeth College Of Pharmacy,
Kolhapur
MATRIX ATTESTED LASER
DESORPTION IONIZATION
TECHNIQUE
1
Guided By
Mr. D. V. Mahuli
Assistant Professor
Department Of Pharmacology

2. Contents
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Introduction
Matrix
Laser
Sample preparation
Mechanism of MALDI
Mass spectrometer
Reproducibility and Performance
Uses of MALDI

3. Introduction
3
Matrix-assisted laser desorption/ionization
Soft ionization technique used in mass spectrometry
Analysis of bio moleculesand large organic molecules
The ionization is triggered by a laserbeam
It is used to determine the molecular weightof Peptides,
Proteins, Antibodies upto size to 300 kDa

4. MALDI is two step process
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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 matrixmaterial. Hot
fumes gets produced during ablation.
Second, the analytemolecules are ionized in the hot
fumes. Ablated species may participate in the ionization of
analyte.

5. Matrix
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Matrix consists of crystallised 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 Matrix material
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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 absorptionin 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

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They typically contain 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
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MALDI techniques typically 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 lasersare used due to
their softer mode of ionization.
IR-MALDIalso 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
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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
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The mechanism of MALDI Done in three steps…
1.Formation of a Solid Solution
2.Matrix Excitation
3.Analyte lonization

11. 1. Formation of solid solution
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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
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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
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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

14. Mechanism of MALDI
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15. Mass Spectrometer
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16. Mass Spectrometer
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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
"ionmirror", deflecting ions with an electric field, thereby
doubling the ion flight path and increasing the resolution.

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Today commercial reflectron TOF instruments reach a
resolving powerm/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) MShas been
demonstrated to be a useful technique where high
resolution MALDI-MS measurements are desired.

18. Reproducibility and Performance
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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 removedby 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
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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
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Will help revolutionize the medical worldand will help
lead to treatments for many diseases
Will be useful for DNA sequencing,thus can be useful
for forensic investigations

21. Conclusion
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Matrix-assisted laser desorption/ionization has become a
gold standard for microbialidentificationin 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 vectorsof human diseases.

22. References
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A text book of Organic spectroscopy by WlliamKemp.
http://www.psrc.usm.edu/mauritz/maldi.html
http://www.psrc.usm.edu/macrog/maldi. htm
Slideshare
https://youtu.be/RuwbeA22rew

23. THANK
YOU
23