Development of methodologies for the analysis of glycans by capillary electrophoresis and mass spectrometry

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Cells make many types of sugars and link them together in diverse combinations to form glycans. Glycans, the most abundant bio-molecules on earth, are an important source of energy and structural materials. Discoveries in the emerging discipline of glycoscience have shown that cells also use glycans to recognize each other and are therefore critical to everything from development to disease. It is the objective of glycoscience to decipher the important biological roles of glycans—these are often facilitated by specific glycan structures. However, the chemical analysis of glycans is a considerable challenge requiring the development of new tools and techniques. The research proposed here seeks to meet this challenge using a combination of instrumental techniques, specifically capillary electrophoresis (CE) and mass spectrometry (MS). I hypothesize that a hyphenated CE-MS technique (a long-term research objective) will be a transformative method of glycan analysis. In support of this objective, new sample preparation and analysis methodologies are required. Glycan analysis by CE requires their prior labelling with fluorescent molecules (fluorophores) to permit sensitive detection, however many of these fluorophores are sub-optimal for MS. Thus new fluorophores suited for both CE and MS will be synthesized and validated. I also propose to develop glycan sample preparation methods that are compatible with these fluorophores. My research will seek to implement these methodologies for the CE and MS analysis of classes of glycans in which the current dearth of robust analytical methods is especially apparent. Specifically, I will focus on three model systems to characterize: (1) the changes in protein-linked glycans during the ripening of cherries, a valuable local (Okanagan) crop; (2) diet-induced changes in gastrointestinal (GI) glycans in mouse models of GI disorders such as colitis; and (3) alterations in pectin, a class of high molecular weight glycan, produced in a collection of flax plants with known mutations in cell wall biosynthetic pathways. In sum, the methods developed through this research program will assist in the efforts of the glycoscience community to structurally characterize glycans; furthermore, the specific systems of proposed study will yield important agriculture, industrial and health benefits. This research will afford students in my lab with excellent training opportunities in modern analytical chemistry, equipping them with research and technical skills that are in high demand in both academia and industry.