The amino acid sequence of ZAP-70 is shown in Table and the list of possible short tryptic peptides of ZAP-70 YY including various phosphorylation states in the kinase domain in Table . For the identification of these and other endogenous phosphopeptides of ZAP-70 digests, proteins were first isolated by gel electrophoresis by cutting the polyacrylamide gel between 50 kDa and 75 kDa followed by in-gel proteolytic digestion as described in the experimental section. The sample complexity and peptide profiles of digested ZAP-70 phosphoproteins from anti-CD3 and anti-CD3+H2O2 stimulated cells were screened using LC-MS/MS (Fig. ). All YY peptides could be identified in the chromatograms i.e. YY (gray, Mw 1302.5 g/mol), YpY (1) and pYY (2) (purple, Mw 1382.5 g/mol) and pYpY (green, Mw 1462.5 g/mol). Confident phosphorylation site localization of isobaric peptides was achieved by comparing their MS/MS fragmentation patterns (Fig. , Fig. ). Thus ALGADDSYpYTAR with characteristic y(4) ion at m/z = 590.2 (Fig. ) and ALGADDSpYYTAR with characteristic y(4) at m/z = 510.2 (Fig. ) verified presence of the pYY and YpY motifs, respectively.