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Mass spectrometry
8/1/03. By Richard Twyman
A high-throughput method for the identification of proteins.
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Mass spectrometry is used for the accurate determination of molecular masses. When applied to proteins, mass spectrometry data can be used to search sequence databases and identify the proteins present in a sample.
Key principles
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Mass spectrometry is most suitable for the analysis of pure proteins or very simple mixtures. The spots excised from two-dimensional gels (see 2D-PAGE) are ideal.
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The protein sample is digested with trypsin, which cuts in a sequence-specific manner to produce a defined set of peptides.
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The masses of the peptides are determined by mass spectrometry.
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The experimentally determined masses are used to search databases of protein sequences.
How does it work?
A mass spectrometer has three components: a source of ions, a mass analyser and a detector. The sample is evaporated in a vacuum and exposed to a high voltage, converting the molecules into gas phase ions. (Special ionisation techniques are required for proteins because they have a low volatility.)
The ions are accelerated through a mass analyser towards a detector. The mass analyser separates the ions according to their mass/charge ratio. The detector records the impact of individual ions, producing peaks on a mass spectrum. The mass of a molecule can be calculated from the mass/charge ratio of its derivative ions.
How is it used?
Mass spectrometry is used to identify and characterise proteins (protein annotation). There are two major approaches.
The first involves digesting a sample with trypsin and determining the masses of the intact peptides, producing a 'peptide mass fingerprint' of a sample. This fingerprint can be used to search protein databases. A search algorithm is used that carries out virtual digests of protein sequences based on the sequence-specificity of trypsin and then calculates the masses of
the predicted peptides from first principles (for example, by adding up the masses of the individual atoms).
If no matches are found, a different procedure called tandem mass spectrometry can be used which results in the random fragmentation of the peptides. The masses of these shorter fragments can be searched against further databases containing short sequences, such as expressed sequence tags. The fragment ions can also be ordered by size and the masses of sequential fragments used
to establish which amino acids have been cleaved off. In this way, the protein sequence can be established and used to search sequence databases to find related rather than exact matches.
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