|dc.description.abstract||The analysis of protein glycosylation is necessary to improve our understanding of various biological processes, as well as to facilitate correlation of glycan structures with healthy and disease states. In contrast to released glycan analysis, the key attractive feature of glycopeptide-based anlaysis is its ability of characterize the glycan population at each glycosylation site on. This dissertation focuses on the development of new approaches for separating, detecting and quantitating glycopeptides using liquid chromatography (LC)-mass spectrometry (MS).
Chapter 1 is an introduction of glycosylation and its biological significant. Also included are description of the two commonly used approaches for glycomic study: released glycan analysis and glycopeptide analysis.
Chapter 2 evaluates hydrophilic interaction chromatography (HILIC) for the separation of isomeric glycopeptide mixtures. The interaction between HILIC and glycopepitdes permit glycopeptides to be resolved based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC is sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 vs α2-6 linkages while these glycans remain attached to peptides.
Chapter 3 investigates the kinetics of glycan release by PNGase F from glycopeptides of IgGs. LC-selective reaction monitoring (SRM) analysis of the glycopeptides from IgGs revealed that slight differences in amino acid sequences were not found to cause a statistically different deglycosylation rate. However, significant differences in the rate constant associated with this reaction were observed between glycopeptides with differing only in glycan structure (i.e. non-fucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc.).
Chapter 4 investigates the PNGase F catalyzed release of glycans from a variety of standard glycoproteins (Ribonuclease B, Fetuin, and Transferrin) to determine if there are correlation between glycan structure, peptide sequence and the rate of glycan release. The experimental results revealed that significant differences in the deglycosylation rate constants were found between glycopeptides with very different peptides sequences but the same glycan structure, as well as between glycopeptides differing only in glycan structures. We predict the differences in release kinetics can lead to significant quantitative variations.||