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. 2014 Aug 28:108:258-68.
doi: 10.1016/j.jprot.2014.05.022. Epub 2014 Jun 4.

Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry

Markus Windwarder  1 , Friedrich Altmann  2
Affiliations

Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry

Markus Windwarder et al. J Proteomics. .

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.

Biological significance: 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.

Keywords: Electron-transfer dissociation; Fetuin; Glycoprotein; O-glycosylation; Phosphorylation.

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