Developing novel mass spectrometry-based approaches to globally investigate protein glycosylation
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Glycosylation is one of the most common and essential protein modifications in cells. It plays a critical role in regulating protein folding, trafficking, and stability, and is involved in a wide range of cellular events such as cell-cell communication, cell proliferation, and immune response. Aberrant glycosylation is closely correlated with different human diseases including cancers and infectious diseases. Glycoproteins in easily accessible bodily fluids including human sera and urine contain plentiful valuable information about the health and disease statuses of an individual, and thus can be used as biomarkers. Meanwhile, antibodies and inhibitors can be designed to specifically target the disease associated glycoproteins for therapeutic treatment. Therefore, comprehensive analysis of protein glycosylation is critical to decipher its biological roles and promote the identification of new biomarkers and drug targets. However, it is extremely challenging to globally analyze glycoproteins in complex biological samples because of the diversity of glycans and low abundance of many glycoproteins. Mass spectrometry (MS)-based proteomics has become a powerful tool for the system-wide identification and quantification of protein glycosylation. This thesis mainly focuses on developing novel and effective MS-based methods for comprehensive studies of protein glycosylation. Chapter 1 describes the overview of comprehensive analysis of protein glycosylation using MS-based proteomics. Chapter 2 illustrates a new method based on vinylboronic acid, which possesses a smaller and more flexible structure compared to commonly used arylboronic acids, for effective enrichment of glycoproteins from complex biological samples. Chapter 3 reports an effective enzymatic oxidation-based approach to comprehensively analyze glycoproteins only located on the cell surface. Chapter 4 describes the large-scale characterization of the cell surface glycoprotein interaction network by integrating chemical crosslinking and MS-based proteomics. Chapter 5 focuses on comprehensive investigation of protein glycation, one type of non-enzymatic glycosylation, with MS-based proteomics. Chapter 6 and Chapter 7 feature the applications of multiplexed quantitative strategies for systematically analyzing the reactivity of tyrosine residues and quantifying fatty acids in human cells. In summary, the work presented in this thesis provides new MS-based approaches and perspectives to globally investigate proteins, especially glycoproteins, and can be extensively applied in biological and biomedical research fields.