Identiﬁcation of c-Src Kinase Substrates research articles Figure 4. MS/MS spectra of novel phosphorylation sites identiﬁed in this study. (A) Phosphopeptide NASTFEDVTQVSSApYQK derived from Cortactin; (B) phosphopeptide TDpYNASVSVPDSSGPER derived from hnRNPK; (C) phosphopeptide QDHPSSMGVpYGQESGG- FSGPGENR derived from Ewing sarcoma breakpoint region 1; (D) phosphopeptide IGGDAGTSLNSNDpYGYGGQK derived from FUSE- binding protein 1; (E) phosphopeptide GPSpYGLSAEVK derived from Calponin-3; (F) phosphopeptide TGAPQpYGSYGTAPVNLNIK derived from FIP1-like 1. identify the phosphopeptides on these proteins which are phosphorylated by c-Src. We systematically designed 312 peptides encompassing all tyrosines from 14 selected proteins (Table 3). These were synthesized in such a fashion that they contained the tyrosine residue being tested in the center. In parallel, an equal number of peptides that have the centric tyrosine residues mutated to phenylalanine were designed. In all, 624 WT or mutant (312 WT and 312 MUT) peptides from 14 proteins were spotted with each sequence being represented in triplicate, on to the glass slides as described earlier.18 The design of these peptide microarrays is analogous to DNA microarrays manufactured by Affymetrix for mRNA expression studies. c-Src kinase assays were performed on the peptide microarrays and the arrays subsequently exposed to phosphorimager screen Journal of Proteome Research Vol. 7, No. 9, 2008 3907