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Title: Glycoproteomic research using mass spectrometry
Author: Bouche, Laura
ISNI:       0000 0004 7657 6591
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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The development of problem-specific mass spectrometric (MS) glycoproteomic strategies has allowed the discovery of previously unknown protein glycosylation in both eukaryotic and prokaryotic organisms. The research in this thesis focuses on the identification and structural characterisation of novel glycan structures in ADAMTS13 and Clostridium difficile. ADAMTS13 is a large multi-domain protein which regulates thrombogenesis by cleavage of the adhesive blood glycoprotein, VWF, so generating smaller less thrombogenic fragments. Glycoproteomic strategies were employed to investigate a secretion-enhancing mutant by comparing the O-glycome of wild type (WT) with synonymous substitution, P118P, and a non-synonymous control, P118F. Identical post-translational modifications (PTMs) but several novel PTMs were discovered in ADAMTS13 including TSR1 O-glycosylation, C-mannosylation of W387 and DiSialyl Core-1 O-glycosylation of S1170. Clostridium difficile is one of the main organisms responsible for morbidity in hospitalised patients, and is the etiological agent of antibiotic-associated diarrhoea and pseudomembranous colitis. The C.difficile cell wall is surrounded by an S-layer composed of two proteins, high molecular weight (HMW) and low molecular weight (LMW) SLPs. 12 slpA gene cassettes have been recently described and cassette-11 carries an insert containing 19 ORFs. Combining different biochemical and ES- and MALDI-MS approaches, including the ETD technique, with genetic experiments, it was demonstrated that LMW SLP in strain Ox247 is glycosylated with a surprisingly large linear pentose-branched oligosaccharide of more than forty sugar residues, and a collaborative NMR study suggests a Phospho- and Acetyl- substituted non-reducing terminal rhamnose. Analysing different C.difficile hypervirulent strains, novel flagellar sulphonated peptidylamido-glycan structures not previously observed in sugar or amino acid chemistry were identified. High resolution mass measurement and negative-ion nanospray MS/MS of cone-voltage-induced fragment ions were crucial in allowing the discovery of a unique terminal Taurine (aminoethyl-sulphonic acid) peptidylamidoglycan unit which could provide a novel strategy to escape the immune system, by the C.difficile becoming more virulent.
Supervisor: Dell, Anne ; Morris, Howard Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral