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J Am Soc Mass Spectrom. 1994 May;5(5):350-8. doi: 10.1016/1044-0305(94)85050-X.

Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry, complementary mobile phases, and sequential exoglycosidase digestion.

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1
Department 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).

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