Site-specific analysis of the O-glycosylation of bovine fetuin by electron-transfer dissociation mass spectrometry.
UNLABELLED
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.
['Windwarder|Markus|M|', 'Altmann|Friedrich|F|']
D000818:Animals D002417:Cattle D060748:Fetuins D006031:Glycosylation D013058:Mass Spectrometry D010455:Peptides D010766:Phosphorylation D011499:Protein Processing, Post-Translational
O-glycosylation Fetuin Electron-transfer dissociation Phosphorylation Glycoprotein