title: Structural elucidation of O-linked glycopeptides by high energy collision-induced dissociation.
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.
authors.affiliation: Department of Pharmaceutical Chemistry and Mass Spectrometry Facility, University of California San Francisco, California, USA.
authors.forename: K F
authors.initials: KF
authors.lastname: Medzihradszkyaff
authors.forename: B L
authors.initials: BL
authors.lastname: Gillece-Castroaff
authors.forename: R R
authors.initials: RR
authors.lastname: Townsendaff
authors.forename: A L
authors.initials: AL
authors.lastname: Burlingameaff
authors.forename: M R
authors.initials: MR
authors.lastname: Hardyaff