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 Application1 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

The mechanism of MALDI

Mass Spectrometer:

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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