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" Targeted Quantitative Proteomic Profiling of Small GTPases in Cultured Cells and Tissues "
Huang, Ming
Wang, Yinsheng
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Latin Dissertation
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English
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899140
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TL0b97b87j
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Chen, Lifan Claire
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Targeted Quantitative Proteomic Profiling of Small GTPases in Cultured Cells and Tissues\ Huang, MingWang, Yinsheng
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2019
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2019
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| Abstract
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Mass spectrometry (MS)-based bottom-up proteomics and quantitative proteomic labeling strategies have led to unprecedented insights into systems biology and provided invaluable resources as a multifaceted analytical tool. We have utilized such techniques to analyze small GTPases of the Ras superfamily, which represent a class of crucial signaling molecules in cells, and the aberrant regulation of their expressions is implicated with various types of human diseases. In this dissertation, I report the development and applications of novel targeted quantitative proteomic methods for high-throughput and reproducible profiling of small GTPases in cultured human cells and patient-derived brain tissues that carry disease-related changes. In Chapter 2, I describe the development of a novel scheduled multiple-reaction monitoring (MRM)-based targeted quantitative proteomic method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC) for the quantification of more than 90 small GTPases in the paired primary/metastatic melanoma cell lines. The data reveal previously unrecognized roles of RAB38 in promoting melanoma metastasis in vitro. In Chapter 3, the established scheduled MRM-based method was further applied to assess the differential expression of small GTPases in wild-type MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method facilitated robust quantification of 96 small GTPases, among which down-regulation of RAB31 was analyzed further and demonstrated to play a role in the development of acquired tamoxifen resistance. In Chapter 4, we extended the use of the scheduled MRM method to comprehensively investigate the differential expression of small GTPases in paired primary/metastatic colorectal cancer cell (CRC) lines SW480 and SW620. With this approach, 83 small GTPases were robustly quantified, leading to the identification of SAR1B as a potential suppressor for CRC metastasis. We also showed that diminished SAR1B expression could stimulate epithelial–mesenchymal transition (EMT), thereby promoting motility and in vitro metastasis of SW480 cells. In Chapter 5, I describe the development of a novel targeted quantitative proteomic assay based on MRM and the use of crude synthetic stable isotope-labeled (SIL) peptides as internal standards (IS) and surrogate standards (SS). By using this approach, we quantified ~80 small GTPases from lysates of frontal cortex from post-mortem Alzheimer’s disease (AD) patient brain tissue samples. The method displayed excellent throughput, sensitivity and reproducibility. Furthermore, we observed that the protein expression levels of Rab3A/C, Rab4A/B and Rab27B proteins, which are involved with synaptic and secretory vesicles, increased with degree of disease severity. The MRM quantification results were further verified by Western blotting.
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Huang, Ming
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UC Riverside
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