title: A novel two-stage tandem mass spectrometry approach and scoring scheme for the identification of O-GlcNAc modified peptides.
abstract: The modification of serine and threonine residues in proteins by a single N-acetylglucosamine (O-GlcNAc) residue is an emerging post-translational modification (PTM) with broad biological implications. However, the systematic or large-scale analysis of this PTM is hampered by several factors, including low stoichiometry and the lability of the O-glycosidic bond during tandem mass spectrometry. Using a library of 72 synthetic glycopeptides, we developed a two-stage tandem MS approach consisting of pulsed Q dissociation (PQD) for O-GlcNAc peptide detection and electron transfer dissociation (ETD) for identification and site localization. Based on a set of O-GlcNAc specific fragment ions, we further developed a score (OScore) that discriminates O-GlcNAc peptide spectra from spectra of unmodified peptides with 95% sensitivity and >99% specificity. Integrating the OScore into the two-stage LC-MS/MS approach detected O-GlcNAc peptides in the low fmol range and at 10-fold better sensitivity than a single data-dependent ETD tandem MS experiment.
authors.affiliation: Department of Proteomics and Bioanalytics, Center of Life and Food Sciences Weihenstephan, Technische Universität München, Emil-Erlenmeyer-Forum 5, 85354 Freising, Germany.
authors.forename: Hannes
authors.initials: H
authors.lastname: Hahne
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D029968:Escherichia coli Proteins; D006020:Glycopeptides / Q000737:chemistry*; D010446:Peptide Fragments; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D038201:alpha-Crystallins / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D006020:Glycopeptides; D010446:Peptide Fragments; D038201:alpha-Crystallins; D000117:Acetylglucosamine