2. CONTENTS:
Introduction
Matrix
Laser
Sample preparation
Mechanism of MALDI
Mass spectrometer
Reproducibility and Performance
Uses of MALDI
3. 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
4. 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 .
5. 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 )
6. 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.
7. 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
8. 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.
10. The mechanism of MALDI
Done in three steps..
(i) Formation of a Solid Solution
(ii) Matrix Excitation
(iii) Analyte Ionization
11. (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.
12. (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.
13. (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
16. 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.
17. 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.
18. 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.
19. 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
20. 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
21. 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