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.
| Residues | Peptide | Site | Observed m/z | Observed mass | Calculated mass | Site assignment |
|---|---|---|---|---|---|---|
| Da | Da | |||||
| O-GlcNAc-modified peptides of rat IRS-1 | ||||||
| 891–915 | pSPGEYVNIEFGSGQPGYLAGPATgSR | 891p, 914g | 1419.2 | 2836.4 | 2837.9 | CID (34) |
| 891–915 | pSPGEYVNIEFGSGQPGYLAGPATSR | 891p | 1318.6 | 2635.2 | 2634.9 | CID |
| 997–1014 | QSYVDTSPVAPVgSYADMR | 1009g | 1095.3 | 2188.6 | 2189.4 | CID/ETD |
| 997–1014 | QSYVDTpSPVAPVSYADMR | 1003p | 1034.4 | 2066.8 | 2066.2 | CID |
| 997–1014 | QSYVDTpSPVAPVgSYADMR | 1003p, 1009g | 1135.0 | 2268.0 | 2269.4 | CID |
| 1039–1055 | SASVpTPQGAAEQAAH | 1045p | 833.1 | 1664.2 | 1662.6 | CID (31) |
| 1027–1073 | TTGAAPPPSgSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR | 1036g | 1133.2 | 4528.8 | 4527.5 | ETD BEMAD (31) |
| 1021–1051 (S1036A) | KVSLPRTTGAAPPPSATASAgSASVTPQGAAE | 1041g | 1028.7 | 3083.1 | 3082.4a | CID |
| O-GlcNAc-modified peptides of human IRS-1 | ||||||
| 981–998 | AVPSSRGDYMTMQMSCPR | 984 or 985g | 1139.95 | 2277.8 | 2278.6 | CID |
| 999–1016 | QSYVDTSPAAPVgSYADMR | 1011g | 1081.7 | 2161.4 | 2060.0 | CID |
| 999–1016 | QSYVDTpSPAAPVSYADMR | 1005p | 1020.1 | 2038.2 | 2038.1 | CID (36) |
| 1029–1074 | ATMAAASSSSAASApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR | 1043p | 1419.3 | 4254.8 | 4255.6 | CID |
| 1029–1074 | ATMAAASSSSAApSApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR | 1041p, 1043p | 1445.4 | 4333.2 | 4335.6 | CID |
| 1029–1074 | ATMAAASSSSAASApSPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR | 1043p, gb | 1485.9 | 4454.7 | 4457.8 | CID |
| 1029–1074 | ATMAAASSSSAASASPTGPQGAAELAAHSSLLGGPQGPGGMSAFTR | gb | 1459.3 | 4374.9 | 4376.8 | CID |
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.