STRUCTURAL ELUCIDATION OF O-LINKED GLYCOPEPTIDES BY HIGH-ENERGY COLLISION-INDUCED DISSOCIATION

O-linked glycopeptides that bear a GalNAc core with and without the pr esence of sialic acid have been analyzed by high energy collision-indu ced dissociation (CID). We show that the CID spectra from the glycosyl ated precursor ions contain sufficient information to identify the pep tide sequence and to determine the glycosylated site(s). Asialo O-link ed 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), whe reas the other exhibited Hex-HexNAc moieties at both Thr(262) and Ser( 264). In addition, sialo and asialo fetuin glycopeptides from a pronas e digest were derivatized with t-butoxycarbonyl-tyrosine, and characte rized by high energy CID analysis. The presence of a Gal beta(1,3)GalN Ac core structure at Ser(264) was confirmed by using the substrate spe cificity of endo-alpha-N-acetylgalactosaminidase. These studies reveal ed the presence of a P-galactosidase specific for beta(1,4) linkages i n the endo-alpha-N-acetylgalactosaminidase preparation employed. Final ly, the relative stability of N- and O-glycosyl bonds to high energy C ID is addressed based upon comparison of the behavior of a synthetic N -linked glycopeptide with analogous O-linked structures.