Mass spectrometry (MS)_Part 3

Matrix Assisted Laser Desorption Ionisation (MALDI)

MALDI is an ionization technique for large and/or labile molecules such as peptides, proteins, polymers, dendrimers, and fullerenes. 

Matrix Assisted Laser Desorption Ionisation (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193) deals well with thermolabile, non-volatile organic compounds especially those of high molecular mass and is used successfully in biochemical areas for the analysis of proteins, peptides, glycoproteins, oligosaccharides, and oligonucleotides. It is relatively straightforward to use and reasonably tolerant to buffers and other additives. The mass accuracy depends on the type and performance of the analyser of the mass spectrometer, but most modern instruments should be capable of measuring masses to within 0.01% of the molecular mass of the sample, at least up to ca. 40,000 Da.

MALDI is based on the bombardment of sample molecules with a laser light to bring about sample ionisation. The sample is pre-mixed with a highly absorbing matrix compound for the most consistent and reliable results, and a low concentration of sample to matrix works best. The matrix transforms the laser energy into excitation energy for the sample, which leads to sputtering of analyte and matrix ions from the surface of the mixture. In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposition. Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.

This technique involves embedding analytes in a matrix which absorbs energy at the wavelength of the laser. The detailed mechanism of the MALDI process, a combination of desorption and ionization, is still being investigated. 

MALDI produces both positive and negative ions, usually in the forms of (M+H)⁺, (M+Na)⁺ and (M-H)^-. The technique also produces multiply charged ions, usually up to +3, as well as dimers, trimers, etc.

Low levels of some salts, buffers, and detergents can be tolerated as well as less than 2% of glycerol. However, data quality and sensitivity may be compromised. Water, acetonitrile, methanol, THF, and other volatile organic solvents can be used. DMSO and DMF cannot be used. Samples should be submitted in small Eppendorf-type tubes.

Sample size depends on molecular weight, the higher the molecular weight the more the sample that is needed. Samples are dissolved in a suitable solvent. Proteins and peptides are usually dissolved in H₂O with 0.1% TFA. 1-10 pmol/µl is needed for MW 1000, 10-50 pmol/µl for MW 20,000, and 100-500 pmol/µl is needed for MW 60-100,000. 

In some cases as little as 250 fmoles of sample is needed on the target. See the table below for the amounts needed for different types of samples. The upper mass limit for MALDI is about 350,000 Da. MALDI is useful for mixtures such as tryptic digests since the technique produces predominantly (M+H)⁺ species for lower molecular weight compounds.

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