Mass spectrometry (MS) has flourished over the last 25 years with the advent of ‘soft’ ionization techniques to measure the molecular masses of proteins and peptides. The two techniques used are electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser-desorption ionization mass spectrometry (MALDI-MS). The main advantage of ESI-MS is the generation of ions in an organic solvent allowing the mass spectrometer to be coupled directly to a HPLC system for on-line analysis during peptide separation. Both techniques allow the accurate mass measurement of intact proteins and the acquisition of sequence information from peptide fragmentation spectra for protein identification.
In conjunction with the growing number of genome sequences that are available in databases, MS can be used to investigate the proteomes of intact cells and subcellular fractions from peptide fragmentation data. This is currently performed either by 1-D or 2-D HPLC-ESI-MS/MS (‘gel-free proteomics’). Moreover, information on changes in protein expression can be obtained by measuring changes in peptide intensities (label-free), by differential isotopic tagging or labelling of peptides (iTRAQ, TMT) or by isotopic labelling of proteins (SILAC) prior to LC-MS/MS. Given the smaller than anticipated number of human genes, functional diversity must be created through other means. Since genome databases provide few clues as to the existence of splice variants of proteins and post-translational modifications (especially phosphorylation, glycosylation and acetylation), one of the great challenges in proteomics in the post-genomic era is to use MS to pin-point these variations and quantify their changes.