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Updated: May 6, 2026
Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases
Published on: December 26, 2011
Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass
K F Medzihradszky1, D A Maltby, S C Hall
1Department of Pharmaceutical Chemistry, Mass Spectrometry Facility, University of California, San Francisco, California, USA.
Abstract:
A strategy for the identification of the site occupancy and glycoform heterogeneity, including sialylation occurring at specific sites of N-linked giycoproteins is presented using the asparagine-linked glycosylation on bovine fetuin for illustration. This is achieved by microbore high-performance liquid chromatography/electrospray ionization mass analysis (LC/ESIMS) of the tryptic glycopeptide mixtures with an acetonitrile-based mobile phase followed by sequential steps of residue (and linkage) specific glycoform degradation and LC/ESIMS analysis at each stage. In addition, chromatographic separation of the site-specific glycoforms of tryptic glycopeptides is accomplished by the use of an alternative, mass spectrometrically compatible mobile phase-water/ethanol/propanol/formic acid. By employing this nontraditional mobile phase for characterizing the complete tryptic digest, and using highly specific exoglycosidases in combination with LC/ESIMS analysis, a previously uncharacterized carbohydrate (a disialo biantennary complex oligosaccharide) was identified as a novel structure at Asn(81) of bovine fetuin. (J Am Sot Mass Spectrom 1994, 5, 350-358).
