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    Deciphering protein glycosylation through novel mass spectrometry-based proteomic strategies

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    XIAO-DISSERTATION-2018.pdf (8.267Mb)
    Date
    2018-04-10
    Author
    Xiao, Haopeng
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    Abstract
    Protein glycosylation is essential for cell survival and proliferation. Comprehensive analysis of protein glycosylation can aid in a better understanding of protein functions, cellular activities, and the molecular mechanisms of diseases. Emerging mass spectrometry (MS)-based proteomics enables comprehensive analysis of protein glycosylation and many other types of modifications. However, due to the heterogeneity of glycans and the low abundance of many glycoproteins in complex biological samples, it is extraordinarily challenging to globally and site-specifically analyze glycoproteins. This thesis focuses on the development of new methods for global analysis of glycoproteins, and the applications of the newly developed methods for biomedical research. This thesis is constituted of six chapters. Chapter 1 is an overview of MS-based glycoproteomics analysis, with an emphasis on the endeavors in the literature to solve the two major problems for global analysis of glycoproteins mentioned above. This chapter retraces the developments of important chemical and enzymatic methods in this field, and includes the discussion regarding how these methods have enabled qualitative and quantitative analyses of glycoproteins in a variety of biological systems. Chapter 2 focuses on the development of a strategy that utilizes the universal recognition between boronic acid and sugars, in order to enrich glycopeptides for LC-MS/MS analysis. Chapter 3 shows the approach of achieving quantitative analysis of protein glycosylation through the combination of boronic acid enrichment and quantitative proteomics. Chapter 4 describes a strategy for cell-surface N-glycoproteome analysis. Metabolic labeling, click chemistry, and MS-based proteomics were combined to specifically map the glycoproteins located only on cell surface. The labeling efficiencies of different sugar analogs were compared, and this method was combined with either stable isotope labeling in cell culture (SILAC) or tandem mass tag (TMT)-labeling to quantitatively study the surface N-glycoproteins. Chapter 5 explains how protein S-GlcNAcylation was unexpectedly found in human cells. Starting with an attempt to profile protein O-GlcNAc, hundreds of S-GlcNAcylation sites were surprisingly identified on cysteine residues. This modification was demonstrated not to be caused by chemical reactions with the cleavable linker during sample preparation nor due to false site assignment. Furthermore, protein S-GlcNAcylation events were investigated with different sugar analog labeling in three cell lines. Chapter 6 features an application of MS-based proteomics in biomedical research. In this chapter, the cellular responses and pleiotropic effects in statin-treated cells on the proteome, glycoproteome, and phosphoproteome levels were analyzed. In addition to the independent projects discussed above, the collaborative projects about that investigation of the cellular mechanisms of gold-nanorod assisted cancer photothermal therapy, and the discordance between mRNA and proteome in ovarian cancer tissues were also conducted. The abstracts of the publications resulted from the collaborations are shown in the appendix. In conclusion, the work presented in this thesis majorly combines chemical biology and modern MS-based proteomics to study protein modifications, especially glycosylation. This thesis strives to advance the techniques of glycoproteomics and apply the state-of-the-art methods to investigate biological and biomedical problems.
    URI
    http://hdl.handle.net/1853/61138
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    • Georgia Tech Theses and Dissertations [23403]
    • School of Chemistry and Biochemistry Theses and Dissertations [1509]

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