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

✍ Scribed by K.F. Medzihradszky; D.A. Maltby; S.C. Hall; C.A. Settineri; A.L. Burlingame

• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 918 KB
• Volume: 5
• Category: Article
• ISSN: 1044-0305
• DOI: 10.1016/1044-0305(94)85050-x

✦ Synopsis

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).