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J Am Soc Mass Spectrom. 1996 Apr;7(4):319-28. doi: 10.1016/1044-0305(95)00682-6.

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

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1
Department of Pharmaceutical Chemistry and Mass Spectrometry Facility, University of California San Francisco, California, USA.

Abstract

O-linked glycopeptides that bear a GalNAc core with and without the presence of 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 Thr(262), whereas the other exhibited Hex-HexNAc moieties at both Thr(262) and Ser(264). 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 Galβ(1,3)GalNAc core structure at Ser(264) was confirmed by using the substrate specificity of endo-α-N-acetylgalactosaminidase. These studies revealed the presence of 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.

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