Strategy for Profiling and Structure Elucidation of Mucin-Type Oligosaccharides by Mass Spectrometry

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Abstract
A strategy combining accurate mass determination, tandem mass spectrometry, structure homology, and exoglycosidases is described that allows the structural characterization of mucin-type O-linked oligosaccharides. The method is used to profile with quantitation the O-linked oligosaccharide (both neutral and anionic) components of the only diploid Xenopus frog, Xenopus tropicalis. Collision-induced dissociation was used to determine connectivity, to identify previously characterized oligosaccharides, and to determine the presence of structural motifs in unknown oligosaccharides. Exoglycosidase digestion was used to identify the individual residues along with the linkages. The enzymes were also used to cleave larger oligosaccharides to smaller units that are similar to previously elucidated components. By using CID, isomeric structures were compared to determine whether they were identical. In this way, the exoglycosidases were more effectively used, and their use was minimized. A total of 35 oligosaccharides including neutral, sialylated, and sulfated were characterized in this way. The relative abundances of all components were also determined based on HPLC.