MALDI (Matrix Assisted Laser Desorption lonization)

o MALDI is also based on "soft ionization" methods where ion formation does not lead
to a significant loss of sample integrity.
o Consequently, the high throughput and speed associated with complete automation
has made MALDI-TOF mass spectrometer an obvious choice for proteomics work on
large-scale.

Advantages
a. Gentle lonization technique
b. High molecular weight analyte can be ionized
c. Molecule need not be volotile
d. Wide array of matrices
e. Produces singly charged ions thus interpretation becomes easy.

fPrior separation by chromatography is not required.

Disadvantages
a. MALDI matrix cluster ions obscure low m/z species (<600)
b. Analyte must have very low vopor pressure
c. Pulsed nature of source limits compatibility with many mass analyzers
d. Coupling MALDI with chromatography can be difficult 27

e. Analytes that absorb the laser can be problematic.

MALDI-Principle
The sample for analysis by MALDI MS is prepared by mixing or coating
with solution of an energy-absorbent, organic compound called matrix.
When the matrix crystallizes on drying, the sample entrapped within the
matrix also co-crystallizes. The sample within the matrix is ionized in an
automated mode with a laser beam. Desorption and ionization with the
laser beam generates singly protonated ions from analytes in the sample.

Matrix and Sample Preparation

o The matrix consists of crystallized molecules, of which the three most
commonly used are - 3,5-dimethoxy-4- hydroxycinnamic acid (sinapinic
acid), a-cyano-4- hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid
(DHB).
o A solution of one of these molecules is made, often in a mixture of highly
purified water and an organic solvent such as acetonitrile (ACN) or ethanol.

o A counter ion source such as Trifiuoroacetic acid (TFA) is usually added to

generate the [M+H] ions.
o A good example ofa matrix solution would be 20 mg/ml sinapinic acid in
ACN: water: TFA (50:50:0.1).
o The matrix performs two important functions:
1. It absorbs photon energy from the laser beam and transfers it into
excitotion energy of the solid system,
2. It serves as a solvent for the analyte, so that the intermolecular forces
are reduced and aggregation of the analyte molecules is held to a
minimum.

o Some desirable characteristics of a typical MALDI matrix are:

a. A strong light absorption property at the wavelength of the laser flux.
b. The ability to form micro-crystals with the sample.
c. A low sublimation temperature, which facilitates the formation of an
instantaneous high-pressure plume of matrix-sample material during the
laser pulse duration.
d. The participation in some kind of a photochemical reaction so that the
sample molecules can be ionized with high yields.
29

MALDI Matrix List

WAVELENGTH
cOMPOUND
OTHER NAMES sOLVENT
(nm)
APPLICATIONS

Acetonitrile, water,
Peptides,
2,5-dihydroxy DHB, Gentisic nucleotides,
benzoic acid acid
methanol, acetone, 337,355, 2bb
oligosaccharides,
chloroform
oligonucleotides

3,5-dimethoxy-4 Sinapic acid,

hydroxycinnamic sinapinic acid; Acetonitrile, water,
chloroform 337,355, 266
337, 355, 266
eptides, proteins,

acid SA
acetone, lipids

a-cyano-4-
hydroxycinnamic CHCA
Acetonitrile, water, 337, 355 Peptides, lipids,
ethanol, acetone nucleotides
acid

Picolinic acid PA Ethanol 266 Oligonucleotides

3-hydroxy picolinic HPA Ethanol 337, 355 Oligonucleotides
acid
 MALDI IONIZATION MECHANISM
laser
University of
analyte/matrix spot
bea
BRISTOL
analyte O Paul J. Gates 2014
ions

To mass
spectrometer

matrix
ions

cation

Sample plate extraction

grid focussing
lens

31

Applications of MALDI
a. Proteomics To identify verify and quantitate: metabolites,
recombinant proteins, proteins isolated from natural
source, peptides and their amino acid sequences.
b. Pharmaceutical i. Bioavailability studies
Analysis i. Drug metabolism studies, pharmacokinetics
ii. Characterization of potential drugs
iv. Drug degradation product analysis
v. Screening of drug candidates
vi. Identifying drug targets

c. Microbiology i. It is used for the identification of microorganisms.

i. Species diagnosis by this procedure is much faster,
more accurate and cheaper than other procedures
based on biochemical tests.

d. Forensic Analysis and i. Pesticides on food

Environmental Analysis i. Soil and groundwater contamination.