Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation |
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Authors: | K F Medzihradszkyaff B L Gillece-Castroaff R R Townsendaff A L Burlingameaff M R Hardyaff |
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Institution: | 1. Department of Pharmaceutical Chemistry and Mass Spectrometry Facility, University of California San Francisco, California, USA 3. Immunogen, Norwood, Massachusetts, USA
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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 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 Galβ(1,3)GalNAc core structure at Ser264 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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