Development And Application Of Mass Spectrometry Methods For Proteomic And Post Translational Modification Analysis

Proteins are essential biomolecules that perform a wide range of biological functions. Post-translational modifications (PTMs) substantially impact protein structure and function, making their characterization essential for understanding complex biological systems. This dissertation focuses on developing and applying novel mass spectrometry (MS)-based methodologies to address challenges in studying two common and important PTMs: phosphorylation and glycosylation. To this end, new enrichment materials and their corresponding workflows, including Cotton Ti-IMAC, epoxy-ATP-Ti4+ IMAC, and Very Weak Anion Exchange (VWAX) have been introduced for efficient phosphopeptide and glycopeptide enrichment. A strategy combining boronic acid enrichment, high-pH fractionation, and EThcD has been developed for comprehensive O-glycosylation profiling. Additionally, the Boost-DiLeu quantitative approach has been introduced to enhance glycopeptide quantification in size-limited samples, while a periodate oxidation-based SUGAR tag labeling method has been established for high-throughput, intact sialylated glycopeptide-specific quantification. These methods have been applied to study human diseases, such as Alzheimer's Disease, providing insights into dysregulated glycosylation patterns and their potential implications in disease pathogenesis. Overall, this work contributes to advancing MS-based proteomics strategies and broadening our understanding of the roles of PTMs in biological systems and is anticipated to inspire future research endeavors in related fields.