Author

He ZhuFollow

Date of Award

12-16-2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Dr. Jun Yin

Second Advisor

Dr. Ning Fang

Third Advisor

Dr. Gangli Wang

Fourth Advisor

Dr. Siming Wang

Abstract

Glycosylation serving as an essential post-translational modification of protein plays an important role in varieties of biological processes including controlling protein folding, regulating immune response, and mediating the interaction between host cells and enzymes, antibodies, receptors, and all kinds of pathogens. The glycosylation is often in low abundance in biological systems, and it exhibits dynamic changes in not only the size and compositions of glycan chains but also the linkages of sialylation. Mass spectrometry is a high-throughput approach and provides a useful tool to decipher the structural details of these dynamic changes in understanding the functions of protein glycosylation. In this dissertation, the glycopeptide-focused and glycan-focused protein glycosylation analysis have been systematically investigated. A set of mass spectrometry-based methods have been established, including glycopeptides enrichment by DEAE-Sepharose coupled with tandem mass spectrometry for the identification of N-glycosylation sites, glycosyltransferase labeling assisted mass spectrometry to distinguish sialylation linkages at the glycopeptide level via the unique reporter fragment ions, and one-pot solid-phase amidation and reductive amination facilitated stable isotope labeling for the quantification of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry. The developed methods have been validated with standard glycoproteins, glycopeptides or glycans, and also been successfully applied in the biological samples analysis, indicating the effective enrichment for glycopeptides, precise identification of linkage-specific sialoglycopeptides, and accurate simultaneous quantification of N-glycosylation between different samples.

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