Sensitive identification and quantification of glycans and proteins derived from biological samples by LC-MS/MS

dc.contributor.committeeChairMechref, Yehia
dc.contributor.committeeMemberPappas, Dimitri
dc.contributor.committeeMemberGamez, Gerardo
dc.creatorYu, Aiying
dc.creator.orcid0000-0002-3323-8184 2022
dc.description.abstractProteins are complex molecules that play many critical roles in biological functions. Protein glycosylation is one of the most common post-translation modifications (PTMs) which involves many biological processes including intercellular signaling, protein stability and localization, inflammation, and immune response. The study of aberrant glycosylation has gained increased attention as it has been correlated with various diseases. However, it remains challenging for glycosylation study due to the low abundance, complex microheterogeneity of glycosylation sites as well as the low ionization efficiency of glycopeptides. Recent advances in sensitive techniques have been developed to facilitate the characterization of glycosylation. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is one of the powerful approaches that have been widely used in the identification and characterization of protein glycosylation. Glycosylation is the process in which glycans were covalently attached to the proteins. Changes in the glycan moieties such as different linkages and glycan isomers can have a strong influence on glycoconjugates which are associated with a variety of human pathologies. Glycomics analysis involves the characterization and quantification of glycans released from glycoproteins. It remains some challenges for comprehensive glycomics analysis including the complexity of glycans, low abundance, and the unstable of the acidic oligosaccharides. To overcome these difficulties, different tandem MS conjugated with advanced separation methods have contributed to the effort. In addition, to stabilize glycan structures, different glycan derivatization methods have been developed. In this dissertation, a reversed-phase LC-MS-based glycomics approach was applied to identify and quantify glycans from biological samples such as kidney brush-border membrane to reveal the way how chronic kidney disease impacts glycosylation. In addition, the permethylation approach was utilized to improve the ionization efficiency of glycans and increase the hydrophobicity of glycans to facilitate separation in reversed-phase chromatography. Through LC-MS-based glycomics analysis, changes of glycan expression such as the altered sialylation and the fucosylation levels were found to be related to chronic kidney diseases. To achieve accurate and high throughput quantification of glycans, we developed a multiplex strategy to incorporate AminoxyTMT and permethylation to derivatize glycans. This strategy enabled up to twelve glycan samples to be analyzed simultaneously which can eliminate deviation from sample preparation and instruments. Other than the glycomic analysis, proteomics analysis was also performed to investigate the correlation of protein expression changes to the different disease statuses. Label-free quantification of proteins was conducted using an LC-MS-based proteomics approach. Through the bottom-up proteomics analysis of kidney brush-border membranes, key proteins that showed significant expression changes between disease-related groups and healthy controls were identified. To further understand the correlation between protein expressions and chronic kidney diseases, pathway analysis was performed to map proteins to biological functions and diseases. The identified key proteins could contribute to chronic kidney diseases through the regulation of some important pathways or impacted by the upstream regulators. In addition, proteome profiling was performed on breast cancer tissues to identify candidate proteins to differentiate four breast cancer subtypes. Unique proteins from each subtype were further investigated to reveal their correlations to the disease functions such as cell proliferation and activation of tumor cell lines. The quantitative proteomics studies might facilitate system biological study and biomarker discovery.
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dc.subjectProtein Expression
dc.subjectChronic Kidney Disease
dc.subjectBreast Cancer
dc.titleSensitive identification and quantification of glycans and proteins derived from biological samples by LC-MS/MS
dc.type.materialtext and Biochemistry Tech University of Philosophy


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