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Title: Enzymatic remodelling of glycans & glycan analysis
Author: Pallister, Edward
ISNI:       0000 0005 0290 8191
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2021
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Glycans are post translational modifications found on proteins and other biomolecules throughout nature. Glycans play a key role in the function of proteins and have been shown to be an important aspect in understanding protein based therapeutic activity in vivo. Enzymes involved in the synthesis of glycans are of significant interest within the biotechnology and biopharma industry given their potential to remodel a glycan's structure and therefore modify therapeutic properties. Analytical techniques such as NMR, ion mobility and mass spectrometry that can quickly analyse glycans and glyco-enzymes are therefore of significant importance. This thesis focuses on addressing some of the outstanding problems in glycan remodelling and glycan analysis. Firstly, this thesis describes how the unique disialyl galactose activity of an α2,6-sialyltransferase from a photobacterium was used to introduce additional sialylation onto the therapeutic glycoprotein α-1-antitrypsin. Analytical approaches required to analyse this unique glycan structure using mass spectrometry were also developed. Secondly, an improved high-throughput analysis of glycans was developed by combining ion-mobility spectrometry (IMS) with a high-throughput mass spectrometry workflow. Unique IMS features allowed for differentiation of isomeric glycans and also glycopeptides in positive ion mode, which previously could not be achieved. Finally, 13C labelled carbohydrates and 13C NMR were used to help characterise two glyco-enzymes, an α2,6 pseudosialidase and also a galactokinase. The 13C labelling and 13C NMR was also useful for reaction monitoring and identifying enzymatic side reactions difficult to detect using other analytical techniques such as mass spectrometry.
Supervisor: Flitsch, Sabine ; Barran, Perdita Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Glycomics ; Sialyltransferase ; Glycan engineering ; Glycans ; mass spectrometry ; Disialylgalactose ; NMR