Mass Spectrometry: Quantitation

graphy (LC) has been used for identification of proteins excised from 1-D or 2-D gels. The automated nature of protein database searching using uninterpreted MS/MS data (4), the low femtomole sensitivity and the ability to identify a protein based on a single MS/MS product ion spectrum allow the identification of more than 100 proteins per day. Thus, extensive protein identifications have been performed for example to identify cancer-related proteins or to study human pathogens. During a study of MHCassociated peptides the use of LC-MS/MS methods for the analysis of complex peptide mixtures was developed. In combination with novel stable isotope-tagging methods (metabolic labeling using amino acids, enzymatic incorporation of O, chemical reactions using ▶ICAT) and multi-dimensional capillary LC (strong cation exchange/reversed phase), tandem mass spectrometry enables quantification of differences in protein expression. In order to study multi-protein complexes, techniques which include LC-MS/MS and tandem affinity purification (TAP) tags for identification of proteins have been established (1). Since MS/MS-generated fragment ion spectra provide a “structural fingerprint” of particular peptides resulting from enzymatic degradation of the protein by specific endoproteinases, MS/MS can be used to determine the site of post-translational modification. Numerous reports show that functionally important modifications such as phosphorylation, glycosylation, methylation, myristoylation and palmitoylation are accessible to analysis via tandem mass spectrometry in such a way that sequences and modified amino acid side chains can be deduced from Cand N-terminal fragment ions of product ion spectra (Fig. 2).