Monday, November 1, 2010

Matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS)

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Matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) is a relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. The ionized biomolecules are accelerated in an elctric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low (10-15 to 10-18 mole) quantities of sample with an accuracy of 0.1 - 0.01 %.
Protein identification by this technique has the advantage of short measuring time (few minutes) and negligible sample consumption (less than 1 pmol) together with additional information on microheterogeneity (e.g. glycosylation) and presence of by-products. Although molecular biology has provided powerful techniques for DNA analysis, this is not yet reflected in protein analysis. Genome sequencing has yielded a wealth of information on predicted gene products, but for the majority of the expressed proteins no function is known. Proteomics is an important new field of study of protein properties (expression levels, interactions, post-translational modifications etc.) and thus can be described as functional genomics at the protein level. The mass accuracy of MALDI-TOF MS will be sufficient to characterise proteins (after tryptic digestion) from completely sequenced genomes (e.g. methanogens, yeast). The use of MALDI-TOF MS for typing of single nucleotide polymorphisms using single nucleotide primer extension has made important progress.
A fully equiped MALDI-TOF MS instrument, a Bruker BIFLEX, is present in our department.

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