Development of Proteomic Strategies to Elucidate Disease Processes in Cancer and Neurological Disorders
Advances in instrumentation and data analysis software have allowed mass spectrometry to become perhaps the most important tool in large scale proteomic studies. Using optimized workflows, it is possible to identify thousands of proteins and protein post-translational modifications in a single mass spectrometry (MS) run from a variety of sample sources, including immortalized cell lines, tissues, and biofluids. However, protein phosphorylation still presents a challenge for analysis because its low abundance and dynamic nature necessitates additional sample preparation steps and special considerations that must be taken into account when quantifying phosphorylation events. Even more difficult is dissection of intricate phosphorylation signaling pathways, which are involved in healthy cellular processes as well as disease pathogenesis. Protein post-translational modifications aside, identification of low abundance proteins from certain sample types can also be problematic. A good example of this is the so-called secretome, or the proteins that are secreted by cells in response to various stimuli. Secreted proteins are released in small amounts into large volumes of complex biofluids or cell culture media, making it difficult to recover these proteins for MS analysis This dissertation discusses strategies for identifying segments of both the phosphoproteome and the secretome. Chapter one is an overview of current phosphoproteomic techniques while chapters two and three highlight the work that has been done to identify the direct substrates of the ABL and CDKL5 kinases, whose dysregulation leads to aberrant phosphorylation signaling and the development of cancer or severe infantile epilepsy, respectively. Chapter four details the development of a novel nanopolymer-based reagent for capture of the secretome from Helicobacter pylori, a pathogen that has been linked to peptic ulcer formation and several types of cancer.
Tao, Purdue University.
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