title: Multiple reaction monitoring mass spectrometry for the discovery and quantification of O-GlcNAc-modified proteins.
abstract: O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3β), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3β is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3β from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3β peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins.
authors.affiliation: Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR) , 20 Biopolis Way, #06-01 Centros, Singapore 138668.
authors.forename: Julien Jean Pierre
authors.initials: JJ
authors.lastname: Maury
authors.forename: Daniel
authors.initials: D
authors.lastname: Ng
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Bi
authors.forename: Muriel
authors.initials: M
authors.lastname: Bardor
authors.forename: Andre Boon-Hwa
authors.initials: AB
authors.lastname: Choo
mesh_terms: D000117:Acetylglucosamine / Q000032:analysis* / Q000235:genetics; D000595:Amino Acid Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis* / Q000235:genetics; D053595:Embryonic Stem Cells / Q000737:chemistry / Q000502:physiology; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000235:genetics
chemical_list: C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D000117:Acetylglucosamine