Table I O-GlcNAc-modified peptides from rat and human IRS-1

Phosphorylated forms of these peptides are also shown. p, phosphorylation, g, O-GlcNAc modification. References previously reporting sites of posttranslational modification are indicated in parentheses. The following chemical modifications occurred during sample preparation (data not shown). Peptides with N-terminal glutamine were partially cyclized to pyroglutamic acid. Peptides were observed with and without oxidation of methionine. Residues 892–922 were observed with +126 Da at Tyr895 most likely due to iodination occurring during alkylation of Cys with iodoacetamide. BEMAD, β-elimination and Michael addition with dithiothreitol.

ResiduesPeptideSiteObserved m/zObserved massCalculated massSite assignment
DaDa
O-GlcNAc-modified peptides of rat IRS-1
    891–915pSPGEYVNIEFGSGQPGYLAGPATgSR891p, 914g1419.22836.42837.9CID (34)
    891–915pSPGEYVNIEFGSGQPGYLAGPATSR891p1318.62635.22634.9CID
    997–1014QSYVDTSPVAPVgSYADMR1009g1095.32188.62189.4CID/ETD
    997–1014QSYVDTpSPVAPVSYADMR1003p1034.42066.82066.2CID
    997–1014QSYVDTpSPVAPVgSYADMR1003p, 1009g1135.02268.02269.4CID
    1039–1055SASVpTPQGAAEQAAH1045p833.11664.21662.6CID (31)
    1027–1073TTGAAPPPSgSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR1036g1133.24528.84527.5ETD BEMAD (31)
    1021–1051 (S1036A)KVSLPRTTGAAPPPSATASAgSASVTPQGAAE1041g1028.73083.13082.4aCID
O-GlcNAc-modified peptides of human IRS-1
    981–998AVPSSRGDYMTMQMSCPR984 or 985g1139.952277.82278.6CID
    999–1016QSYVDTSPAAPVgSYADMR1011g1081.72161.42060.0CID
    999–1016QSYVDTpSPAAPVSYADMR1005p1020.12038.22038.1CID (36)
    1029–1074ATMAAASSSSAASApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR1043p1419.34254.84255.6CID
    1029–1074ATMAAASSSSAApSApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR1041p, 1043p1445.44333.24335.6CID
    1029–1074ATMAAASSSSAASApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR1043p, gb1485.94454.74457.8CID
    1029–1074ATMAAASSSSAASASPTGPQGAAELAAHSSLLGGPQGPGGMSAFTRgb1459.34374.94376.8CID
  • a Expected mass of peptide 1021–1051 following site-directed mutagenesis of S1036A.

  • b Indicates peptides in which the site(s) of O-GlcNAc modification could not be assigned.

    O-GlcNAc modification occurred at either 984 or 985, residues underlined.