Extended Sialylated O-Glycan Repertoire of Human Urinary Glycoproteins Discovered and Characterized Using Electron-Transfer/Higher-Energy Collision Dissociation.

ABSTRACT

A relatively novel activation technique, electron-transfer/higher-energy collision dissociation (EThcD) was used in the LC-MS/MS analysis of tryptic glycopeptides enriched with wheat germ agglutinin from human urine samples. We focused on the characterization of mucin-type O-glycopeptides. EThcD in a single spectrum provided information on both the peptide modified and the glycan carried. Unexpectedly, glycan oxonium ions indicated the presence of O-acetyl, and even O-diacetyl-sialic acids. B and Y fragment ions revealed that (i) in core 1 structures the Gal residue featured the O-acetyl-sialic acid, when there was only one in the glycan; (ii) several glycopeptides featured core 1 glycans with disialic acids, in certain instances O-acetylated; (iii) the disialic acid was linked to the GalNAc residue whatever the degree of O-acetylation; (iv) core 2 isomers with a single O-acetyl-sialic acid were chromatographically resolved. Glycan fragmentation also helped to decipher additional core 2 oligosaccharides a LacdiNAc-like structure, glycans carrying sialyl LewisX/A at different stages of O-acetylation, and blood antigens. A sialo core 3 structure was also identified. We believe this is the first study when such structures were characterized from a very complex mixture and were linked not only to a specific protein, but also the sites of modifications have been determined.