Integrated glycomics, proteomics, and glycoproteomics of human leukocytes and glioblastoma tissue microarrays
This thesis includes studies on N-, mucin type O-, and glycosaminoglycan (GAG)-linked glycosylation in human biospecimens. Glycosylation plays a central role in biological processes, including protein folding, immune surveillance, and regulation of cell growth. The structures of GAG are regulated in a tissue-specific manner. Heparan sulfate (HS) and chondroitin sulfate (CS) are the two types of GAGs targeted in this thesis. Human leukocytes express both CS and HS GAGs with CS being the more abundant type; however, little is known regarding the properties and structures of GAG chains, their ranges of variability among normal subjects, and changes in structure associated with disease conditions. We measured the relative and absolute disaccharides abundances of HS and CS for purified B, T, NK cells, monocytes, and polymorphonuclear leukocytes (PMNs) using size exclusion chromatography-mass spectrometry (SEC-MS). We found that all leukocytes express HS chains with levels of sulfation more similar to heparin than to organ-derived HS. In addition, CS abundances varied considerably in a leukocyte cell type specific manner. Therefore, our results established the ranges of GAG structures expressed on normal leukocytes as well as necessary for subsequent inquiry into disease conditions. Glioblastoma (GBM) accounts for 30% of human primary brain tumors. It is deadly and highly invasive. In past decades, most GBM research focused on pathophysiological changes in genome. There remains a dearth of knowledge regarding alterations in glycomics, glycoproteomics, and proteomics during GBM tumorigenesis. Therefore, we developed a comprehensive platform for high-throughput sample preparation with surface digestion for tissue microarrays, LC-MS/MS data dependent acquisition, and semi-automated data analysis to integrate glycomics, glycoproteomics, and proteomics for different grade of tumor and different subtypes of GBM. By analyzing GBM tissue microarrays, we found tumor grade and subtype specific changes to the expression of biomolecules. We also identified approximately 100 site-specific N- and mucin type O-glycosylations, the majority of which were previously unreported. Overall, our results improved the fundamental understandings about GBM pathogenesis.