Fig. 2.

Phosphorylation and O-GlcNAc modification of human IRS-1 residues 1029–1074. A, CID MS/MS of the triply charged precursor ion at m/z 1459.3 is consistent with O-GlcNAc modification of the peptide 1029–1074. The calculated and observed molecular masses of the O-GlcNAc-modified peptide were 4376.8 and 4374.9 Da, respectively. Neutral loss of N-acetylglucosamine (68.3) from the triply charged precursor at m/z 1459.3 yielded the most abundant ion at m/z 1391.5. Complete neutral loss of the GlcNAc from fragment ions precluded determination of the site of modification. The b and y ions are labeled according to the unmodified peptide. Ions retaining the GlcNAc modification are indicated with an asterisk. B, CID MS/MS of the triply charged precursor ion at m/z 1419.3 is consistent with phosphorylation of peptide 1029–1074 at Ser¹⁰⁴³. The calculated and observed molecular masses of the phosphopeptide were 4255.6 and 4254.8 Da, respectively. b and y ions are labeled according to phosphorylation at Ser¹⁰⁴³. C, CID MS/MS of the phosphorylated and O-GlcNAc-modified peptide 1029–1074 further confirms phosphorylation at Ser¹⁰⁴³. The calculated and observed molecular masses of the phosphorylated and O-GlcNAc-modified peptide were 4457.8 and 4454.7 Da, respectively. Ions retaining the GlcNAc modification are indicated with an asterisk. D, MS/MS of the triply charged precursor at m/z 1445.4 is consistent with phosphorylation at Ser¹⁰⁴¹ and Ser¹⁰⁴³. The calculated and observed molecular masses of the phosphorylated peptide were 4335.6 and 4333.2 Da, respectively. Within the peptide sequence, the sites of phosphorylation are indicated with a “p.”