DEVELOPMENT OF QUANTITATIVE PROTEOMIC STRATEGIES TO IDENTIFY TYROSINE PHOSPHATASE SUBSTRATES

Protein tyrosine phosphorylation is an essential posttranslational modification that controls cell signaling involving various biological processes, including cell growth, proliferation, migration, survival, and death. Balancing tyrosine phosphorylation levels is necessary for normal and pathological states, and this reversible mechanism occurs through protein tyrosine kinases and phosphatases. Advancements in instrumentation and applying conventional biochemical and genetic methods have led to cell signaling studies and pharmaceutical development discoveries. However, there is still a lack of understanding of tyrosine phosphatases' mechanisms, substrates, and activities within complex networks. The challenges remain in the tyrosine phosphatase field due to the low abundance and dynamic nature, sample preparation steps, and sensitivity to detect tyrosine phosphorylation events. Although mass spectrometry (MS)-based phosphoproteomics has allowed the identification of thousands of phosphotyrosine sites in a single run, protein phosphorylation poses another analysis caveat of dissecting complex phosphorylation signaling pathways involved in healthy cellular processes similarly in disease pathogenesis. This dissertation discusses strategies for improving tyrosine phosphatase sample preparation and identifying the tyrosine phosphatases' direct substrates. Chapter one is an overview of current techniques to study tyrosine phosphatases. In contrast, chapters two and three highlight the work that has been done to identify the direct substrates of phosphatase SHP2 and PTP1B, respectively, whose dysregulation leads to the development of cancers. In chapter 2, we describe a novel method that incorporated three separate MS-based experiments to identify the direct substrates of phosphatase SHP2: immunoprecipitation of substrate trapping mutants complex, in vivo global phosphoproteomics, and in vitro dephosphorylation of SHP2 phosphatase substrates. With immunoprecipitation of substrate trapping mutant experiment, weak and ...