Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation

K. F. Medzihradszky, B. L. Gillece-Castro, R. R. Townsend, A. L. Burlingame, M. R. Hardy

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

O-linked glycopeptides that bear a GalNAc core with and without the presence ot sialic acid have been analyzed by high energy collision-induced dissociation (CID). We show that the CID spectra from the glycosylated precursor ions contain sufficient information to identify the peptide sequence and to determine the glycosylated site(s). Asialo O-linked glycopeptides, previously prepared from a tryptic digest of bovine fetuin were studied. One of the glycopeptides contained only a single Hex (hexose)-HexNAc (N-acetylhexosamine) substitution at Thr262, whereas the other exhibited Hex-HexNAc moieties at both Thr262 and Ser264. In addition, sialo and asialo fetuin glycopeptides from a pronase digest were derivatized with t-butoxycarbonyl-tyrosine, and characterized by high energy CID analysis. The presence of a Gal β(1,3)GalNAc core structure at Ser264 was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence ot a β-galactosidase specific for β(1,4) linkages in the endo-α-N-acetylgalactosaminidase preparation employed. Finally, the relative stability of N- and O-glycosyl bonds to high energy CID is addressed based upon comparison of the behavior of a synthetic N-linked glycopeptide with analogous O-linked structures.

Original languageEnglish
Pages (from-to)319-328
Number of pages10
JournalJournal of the American Society for Mass Spectrometry
Volume7
Issue number4
DOIs
StatePublished - Apr 1996

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