title: Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry.
abstract: Bovine fetuin often finds use as a test model for analytical methods, but the exact occupancy of its O-glycosylation sites has not yet been determined. An obstacle for a closer inspection of the five or six O-glycosylation sites is the close spacing of several sites on the same tryptic peptide. The advent of ion-trap instruments with electron-transfer dissociation (ETD) capability and - for the type of instrument - high resolution prompted us to probe this technology for the investigation of the intricate posttranslational modifications O-glycosylation and phosphorylation. Much information could be obtained by direct-infusion ETD analysis of the fully sialylated tryptic 61-residue peptide harboring 8 hydroxyl amino acids of which four were indeed found to be, if only partially, glycosylated. The middle-down approach allowed recognizing an order of action of O-GalNAc transferase(s). No such hierarchy could be observed for phosphorylation. ETD fragmentation on an ion trap thus allowed in-depth analysis of a large, multiply O-glycosylated peptide, however, only by data accumulation over several minutes by direct infusion of a prefractionated sample. O-glycosylation and phosphorylation sites re-defined and their occupancy including that of N-glycans were defined by this study.
  
  
  CONCLUSIONS
  O-glycosylation of natural or recombinant proteins poses a challenge because of the lack of unambiguous consensus sites, the agglomeration of several O-glycans in close proximity and the lack of efficient O-glycosidases. Even bovine fetuin, a frequently used test glycoprotein for glycosylation analysis, has hitherto not been fully characterized in terms of site occupancy. This gap shall hereby be closed by application of electron-transfer dissociation mass spectroscopy.
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU), Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Windwarder
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Vienna (BOKU), Austria. Electronic address: friedrich.altmann@boku.ac.at.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
mesh_terms: D000818:Animals; D002417:Cattle; D060748:Fetuins / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
chemical_list: D060748:Fetuins; D010455:Peptides
keywords: Electron-transfer dissociation; Fetuin; Glycoprotein; O-glycosylation; Phosphorylation