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.
Highlights
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
The present study examined the use of data-dependent neutral loss MS3 mass spectrometry and chemical derivatization prior to mass spectrometry to identify novel sites of O-GlcNAc modification of IRS-1
Trypsin-digested samples were analyzed by LC-MS/MS using data-dependent neutral loss MS3 mass spectrometry
Summary
Protein Standards—A synthetic O-GlcNAc-modified peptide, PSVPVS(O-GlcNAc)GSAPGR, derived from UL32 protein of human cytomegalovirus was a gift from Dr G. Mixtures of protein standards spiked with the synthetic O-GlcNAcmodified peptide were dried, oxidized with performic acid (PFA) vapor in a vacuum chamber containing 1 ml of PFA for 1 h at room temperature as described by McLachlin and Chait [30], and digested with trypsin (Promega) overnight at 37 °C. Chemical Derivatization of O-GlcNAc-modified Peptides with DTT—Peptides that had been oxidized and digested were subjected to BEMAD for the removal of the N-acetylglucosamine from Ser/Thr residues and replacement with DTT [21]. For the analysis of DTT-derivatized peptides of IRS-1, the mass spectrometer was operated in data-dependent mode with one survey MS scan followed by five MS/MS scans on the five most intense ions. Blots were stripped and reprobed with the S-protein conjugated to HRP (Novagen)
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