title: Quantification of post-translationally modified peptides of bovine alpha-crystallin using tandem mass tags and electron transfer dissociation.
abstract: The modification of Ser/Thr residues in proteins by addition of single O-linked N-acetylglucosamine (O-GlcNAc) moieties play an important role in cell regulation. However, understanding the cellular mechanisms that regulate O-GlcNAc glycosylation has been challenging due to the difficulty in detection and quantification of this modification. Mass spectrometry-based multiplex quantitative approaches have been successfully employed to measure relative phosphorylation levels using collisionally induced dissociation (CID). However, labile modifications such as O-GlcNAc are lost prior to fragmentation of the peptide backbone in conventional CID, often preventing correct peptide identification, localization of the modified site, and as a result, relative quantification. Compared to CID, Electron Transfer Dissociation (ETD) preserves labile post-translational modifications (PTMs), and allows direct mapping of peptide/protein modifications. This is the first report to assess the utility of combining multiplexed isobaric tandem mass tag (TMT) labeling and ETD for relative quantification of labile PTMs. ETD analysis of both labeled and unlabeled peptides from bovine alpha-crystallins pinpointed at least one O-GlcNAc containing modification site in each of the protein subunits, in addition to a multitude of other PTMs, including glycation, phosphorylation, and acetylation. Moreover, ETD of TMT(6) labeled peptides produced four unique reporter ions that could be used for relative quantification. TMT reporter ion ratios measured by ETD had similar accuracy and precision as those obtained by conventional CID techniques. When applied to glycosylated or otherwise modified peptides, ETD was the only dissociation method which consistently provided confident sequence identification, PTM localization, and quantitative information, all in the same spectrum. This suggests that ETD-based workflows can be complementary to traditional CID approaches when used for simultaneous qualitative and quantitative analysis of modified peptides.
authors.affiliation: Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA.
authors.forename: Rosa I
authors.initials: RI
authors.lastname: Viner
authors.forename: Terry
authors.initials: T
authors.lastname: Zhang
authors.forename: Tonya
authors.initials: T
authors.lastname: Second
authors.forename: Vlad
authors.initials: V
authors.lastname: Zabrouskov
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D004583:Electrons; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D038201:alpha-Crystallins / Q000737:chemistry*
chemical_list: D010455:Peptides; D038201:alpha-Crystallins