UC Davis

Glycans are critical cell components due to their remarkable structural, modulatory, and biological functions. Despite the recent advancements in glycomic and glycoproteomic techniques, limitations still exist in elucidating specific glycan phenotypes and glycan-protein interactions in living systems. This dissertation describes the development and application of novel mass spectrometry-based methods to characterize the phenotypes of cell glycogen as well as the protein interactions of cell-surface fucose and sialic acid.Chapter I provides an overview of the structures, functions, and interactions of glycans. It further describes the current mass spectrometry approaches for glycan profiling and quantitation as well as for characterizing glycan-protein interactions. Chapter II demonstrates an extended application of our carbohydrate quantitation and structural analysis methods to characterize cell glycogen phenotypes using Hep G2, a human liver cancer cell line. It reports on the change of cell glycogen storage and degree of branching under the influence of insulin treatments. Chapter III presents a new proximity labeling methodology, named protein oxidation of fucose environments, to identify potential cell-membrane fucose-binding protein candidates and their spatial arrangement around the fucose. Chapter IV introduces a glycoprotein-protein cross-linking platform to reveal the cell-surface protein networks mediated by sialic acids. The method provides detailed information about the interacting glycoproteins and proteins, including the glycoforms and glycosites in addition to the sequences. Chapter V presents an expansion of the cross-linking platform with the development of enrichable and cleavable chemoproteomic reagents and the corresponding workflows. The methods enable the enrichment of cross-linked products using biotin-streptavidin interactions and thus, offer a sensitive approach suited for detection of low-abundance glycan-protein interactions. The content of Chapter IV has been published in Chem. Sci., 2021, 12, 8767-8777 and the contents of Chapters II, III, and V are in preparation for manuscript submission at the time when this dissertation is written. Co-authors who contributed to the research and manuscripts include Michael Russelle Alvarez, Yasmine Bouchibti, Vincent Chang, Ye Chen, Carlito Lebrilla, Qiongyu Li, Emanual Maverakis, Joeriggo Reyes, Ying Sheng, Kemal Solakyildirim, Yixuan Xie, and Gege Xu (in alphabetical order). This dissertation would not have been possible without their contribution.