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. 2006 Feb;5(2):313-23.
doi: 10.1074/mcp.M500314-MCP200. Epub 2005 Oct 22.

Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1

Lauren E Ball  1 , Mary N Berkaw, Maria G Buse
Affiliations

Identification of the major site of O-linked beta-N-acetylglucosamine modification in the C terminus of insulin receptor substrate-1

Lauren E Ball et al. Mol Cell Proteomics. 2006 Feb.

Abstract

Signal transduction from the insulin receptor to downstream effectors is attenuated by phosphorylation at a number of Ser/Thr residues of insulin receptor substrate-1 (IRS-1) resulting in resistance to insulin action, the hallmark of type II diabetes. Ser/Thr residues can also be reversibly glycosylated by O-linked beta-N-acetylglucosamine (O-GlcNAc) monosaccharide, a dynamic posttranslational modification that offers an alternative means of protein regulation to phosphorylation. To identify sites of O-GlcNAc modification in IRS-1, recombinant rat IRS-1 isolated from HEK293 cells was analyzed by two complementary mass spectrometric methods. Using data-dependent neutral loss MS3 mass spectrometry, MS/MS data were scanned for peptides that exhibited a neutral loss corresponding to the mass of N-acetylglucosamine upon dissociation in an ion trap. This methodology provided sequence coverage of 84% of the protein, permitted identification of a novel site of phosphorylation at Thr-1045, and facilitated the detection of an O-GlcNAc-modified peptide of IRS-1 at residues 1027-1073. The level of O-GlcNAc modification of this peptide increased when cells were grown under conditions of high glucose with or without chronic insulin stimulation or in the presence of an inhibitor of the O-GlcNAcase enzyme. To map the exact site of O-GlcNAc modification, IRS-1 peptides were chemically derivatized with dithiothreitol following beta-elimination and Michael addition prior to LC-MS/MS. This approach revealed Ser-1036 as the site of O-GlcNAc modification. Site-directed mutagenesis and Western blotting with an anti-O-GlcNAc antibody suggested that Ser-1036 is the major site of O-GlcNAc modification of IRS-1. Identification of this site will facilitate exploring the biological significance of the O-GlcNAc modification.

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Figures

Fig. 1
Fig. 1. LC-MS/MS of trypsin-digested IRS-1 peptide 1027−1073 with and without O-GlcNAc modification
A, extracted ion chromatograms showing the elution profiles of residues 1027−1073 with (m/z 1510.2) and without (m/z 1442.6) O-GlcNAc modification. B, mass spectrum acquired at 33−33.2 min. C, MS/MS of precursor at m/z 1442.5 confirming the identity of the peptide 1027−1073 (TTGAAPPPSSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR). D, MS/MS of O-GlcNAcylated precursor at m/z 1510.3 confirmed the sequence of 1027−1073 and shows loss of the monosaccharide upon CID. Generation of the neutral loss ion at 1442.6 triggered MS3 and facilitated detection of this peptide. The expected mass shift due to O-GlcNAc modification is 203.2 Da. Fragment ions in both MS/MS spectra are labeled according the predicted b and y ions of the unmodified peptide.
Fig. 2
Fig. 2. LC-MS/MS of phosphopeptide 1039−1055 (SASASVpTPQGAAEQAAH where pT is phosphothreonine)
MS/MS of the doubly charged precursor ion at m/z 833.1 revealed Thr-1045 as the site of phosphorylation. The asterisks indicate ions that exhibited neutral loss of phosphoric acid (98 Da).
Fig. 3
Fig. 3. MALDI-TOF MS of IRS-1 tryptic peptides before and after β-elimination/derivatization with DTT and after thiol chromatography
The insets highlight mass range 4200−5000. A, trypsin digest of oxidized, alkaline phosphatase-treated IRS-1. The average calculated [M + H]+ of the oxidized peptide 1027−1073 with and without O-GlcNAc modification is 4559.8 and 4356.7, respectively. B, IRS-1 peptides following β-elimination and Michael addition with DTT. The average calculated [M + H]+ of oxidized, DTT-derivatized 1027−1073 is 4492.0. C, sulfhydryl-reactive peptides purified by thiol chromatography. Masses that correspond to predicted DTT-derivatized peptides of IRS-1 are indicated by asterisks and are listed in Table II. The expected increases in mass due to modification by N-acetylglucosamine or derivatization with DTT are 203.2 and 136.2 Da, respectively.
Fig. 4
Fig. 4. MS/MS of the DTT-derivatized peptide 1027−1073 precursor ion at m/z 1498.0 (3+)
The fragmentation pattern is consistent with DTT modification at Ser-1036 (TTGAAPPPSSTASASASVTPQGAAEQAAHSSLLGGPQGPGGMSAFTR).
Fig. 5
Fig. 5. O-GlcNAc modification of recombinant wild type and mutant IRS-1 (IRS-1 AAA)
Wild type (wt) or mutant (AAA) IRS-1 proteins were expressed in HEK293 cells treated for 18 h with 50 μm PUGNAc to increase the level of protein O-GlcNAc modification. Nickel affinity-purified protein was separated by SDS-PAGE and transferred to nitrocellulose. The presence of O-GlcNAc modification was assessed by probing with an anti-O-GlcNAc antibody. Total IRS-1 protein loaded was assessed by probing the stripped blot with the S-protein-HRP conjugate. WB, Western blot.
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