Novel mass spectrometry-based methods to identify and quantify extracellular glycoproteins
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Extracellular glycoproteins are extremely important to a variety of biological processes, including immune response, cell interactions and disease development. Despite their critical importance, glycoproteins are challenging to analyze due to their low abundance and heterogeneity of glycans. The work in this dissertation focuses on the development of mass spectrometry-based methods to globally analyze cell surface and secreted glycoproteins. First, a novel method was developed for site-specific identification of the cell surface N-glycoproteome in HEK 293T cells. This method was applied to investigate cell surface glycoprotein changes in MCF 10A cells undergoing the epithelial-mesenchymal transition. Next, secreted proteins and glycoproteins were comprehensively analyzed from Saccharomyces cerevisiae, which is an excellent model system for eukaryotic cells. Finally, an enhanced digestion method for improved membrane protein identification by MS was developed for applications with membrane glycoprotein analysis. Altogether, this work affords new opportunities and methods for large-scale analysis of extracellular glycoproteins, which can be extensively applied to further decode the extracellular glycoproteome.