Virtually all eukaryotic proteins are covalently modified in some way during or after translation. Another focus of our work is to identify posttranslational modifications on proteins and to examine the role of these modifications on the functioning of the proteins. The modifications can be characterized by comparing the accurate mass of a modified protein or peptide with the predicted mass based on its amino acid sequence. Any discrepancies may correspond to posttranslational modifications. An example of a common posttranslational modification with an important role in the regulation of protein activities is phosphorylation on serine, threonine or tyrosine residues. We have established a useful method for identifying potential phosphopeptides present at low levels in complex mixtures of nonphosphorylated peptides by comparison of MALDI-TOF mass spectra taken in both negative and positive ion modes. Putative phosphorylated peptides are then sequenced in the Q-TOF mass spectrometer to confirm phosphorylation and to identify the phosphorylated amino acid. We have also developed or implemented techniques to identify other modifications such as proteolysis, glycosylation, and lipidation of proteins.

MALDI Q-TOF MS of 10% of the methylated tryptic peptides from the in-gel digestion of 1 pmol of autophosphorylated FGFR2 kinase domain operated in positive (a) and negative (b) modes. The peaks with mass difference of 80 in b indicate different levels of phosphorylation (e.g. peaks of m/z 1717.74, 1797.71, and 1877.71 are the mono-, di-, and triphosphorylated forms of the peptide 580–592). a, inset, shows the MS/MS spectrum of the diphosphorylated species of peptide 650–659 mDINNImDpYpYKmK, M+H+ = 1487.63. c, MS/MS spectrum of the nonmethylated monophosphorylated peptide of sequence RPPGMEYpSYDINR from the activation loop of the FGFR2 kinase insert region.

Image Adapted from Molecular & Cellular Proteomics 4:809-818, 2005