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Evaluation and development of precise methods for glycan analysis using mass spectrometry

University of British Columbia

Glycosylation, which is covalent addition of carbohydrate residue(s) onto biomolecule(s), is one of the post-translational modifications present of proteins which helps in expansion of the information content present within the cells; protein-linked carbohydrates are called glycans. The glycome, defined as the entire composition of glycans inside an organism, is dynamic and dependent on cellular microenvironment and physiological conditions. At times of cellular stress or infections, the glycome of an organism often changes in response, making these posttranslational modifications important bio-markers of numerous diseases and disorders. In this thesis, we present the glycomic profile of 16 different chicken duodenum mucus samples collected from birds that had been subjected to stress and/or infection with an increasingly problematic gastrointestinal (GI) pathobiont Clostridium perfringens. These glycomic analyses were conducted using liquid chromatography coupled mass spectrometry (LC-MS) in order to better understand how C. perfingens deploys its carbohydrate-active enzymes in order to establish devastating GI infections in poultry. This glycomic study prompted a more detailed investigation into problems associated with LC-MS-based quantitation of glycans and several different ways in which these may be mitigated by data normalization methods. It was discovered that both sample preparation and data normalization methods have a substantial, and variable impact on the precision of glycomic methods. On the basis of these studies, a new chemical approach to producing stable isotope-labelled glycan internal standards was devised and evaluated, which has been dubbed D3-tagging, with D3 referring to three deuteria atoms present on N-acetylated carbohydrates. The D3-tagged internal standards were found to improve the precision of glycomics data, but at the expense of analytical accuracy.

Text

2021-01-08

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/77031

Chemistry

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/