N- and O- linked Glycosylation, Developing Mass Spectrometric Strategies for the Characterisation of Glyco-epitopes
Kenny, Diarmuid T
MetadataShow full item record
This item's downloads: 1805 (view details)
The overall goal of this thesis was to adopt a liquid chromatography-mass spectrometry (LC-MS) platform for glycomic analysis of glycoproteins. In particular we were interested in characterizing complex glycomic samples such as membrane associated N-linked oligosaccharides and the mucin O- linked oligosaccharides with the aim of identifying particular glycosylation traits or glyco-epitopes that could have biological significance. For the manual interpretation of MSn spectra of oligosaccharides, the acquisition of information-rich MSn spectra is essential. The rearrangement of fragment ions during collision induced dissociation (CID) can complicate the interpretation of the MSn and result in the mis-assignment of an oligosaccharide structure. We have shown that the migration of sulfate groups can occur when sulfated oligosaccharides are fragmented by CID in negative ion mode. The migration is promoted by the presence of a mobile proton and the steric availability of the sulfate groups. Fragmentation by high energy C-trap dissociation (HCD) limits the migration of sulfated residues to where it cannot be detected. Our analysis of membrane associated N-linked oligosaccharides showed that the use of semi-quantitative data can be useful tool at highlighting difference in glycosylation and the use of statistical tools such as monosaccharide composition analysis (MSAC) was useful at providing a global overview of the glycosylation or sorting the oligosaccharide according to particular glycosylation traits such as common core extensions or terminal epitopes provided information on the biological aspects of the oligosaccharides. Our characterisation of O-linked oligosaccharides derived from MUC5AC of healthy and tumour-associated tissue revealed the presence of the novel lacdiNAc (GalNAc Beta 1-4GlcNAc) epitope on gastric mucin. We identified the lacdiNAc epitope by comparing the MS3 of the lacdiNAc containing structure to known standards with terminal N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) residues and by digestion of the lacdiNAc containing oligosaccharides with exo-N-hexosaminidase. We believe this epitope plays a role in inhibiting the binding and proliferation of Helicobacter pylori to MUC5AC by limiting the synthesis of oligosaccharides displaying Leb and sLex epitopes which are required for the adhesion of H. pylori via its adhesins BabA and SabA respectively.
This item is available under the Attribution-NonCommercial-NoDerivs 3.0 Ireland. No item may be reproduced for commercial purposes. Please refer to the publisher's URL where this is made available, or to notes contained in the item itself. Other terms may apply.
The following license files are associated with this item: