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From Discovery-Based to Targeted Proteomics


Mass spectrometry-based proteomic approaches have become the method of choice for performing discovery-based quantitative global proteomic analysis, as well as targeted proteomics for the reproducible analysis of a selected set of proteins. In this dissertation, we reported the application of these approaches in the profiling of global proteome with discovery-based proteomics approach, and profiling of global kinome with targeted proteomic analysis.

In Chapter two, we studied hexavalent chromium [Cr(VI)]-induced alteration of proteomic landscape in human skin fibroblast cells. We utilized MS-based quantitative approach for assessing the global proteome alteration in response to Cr(VI) treatment. Here with the use of in-gel digestion and mass spectrometry analysis in data-dependant mode, we were able to quantify ~4600 unique proteins, among which around 10% exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 uM Cr(VI). From these results, we revealed that Cr(VI) induced its cytotoxic effect via activating the cholesterol biosynthesis pathway.

In Chapter three, we combined the filter-aided sample preparation method with LC-MS/MS analysis to reveal monomethylarsonous acid [MMA(III)]-induced alteration in the protein expression level in human skin fibroblasts. From our study, among the ~6500 quantified unique proteins, ~300 displayed significant changes in expression after exposure with 2 uM MMA(III) for 24 h. Subsequent analysis revealed the inhibition of de novo cholesterol biosynthesis upon MMA(III) treatment.

In Chapter four, we applied adenosine triphosphate (ATP) affinity probe and scheduled multiple-reaction monitoring analysis for profiling global kinome in human cells in response to arsenite treatment. From the quantification of ~250 kinases, we found the expression of several kinases involved in cell cycle progression was changed significantly upon arsenite treatment. The up-regulation of cyclin-dependant kinase 1 was further verified.

In Chapter five, we utilized stable isotope-labeled ATP affinity probe in conjunction with scheduled MRM analysis for profiling global kinome signatures of the radioresistant MCF-7/C6 breast cancer cells. We rigorously quantified 120 kinases, of which one third exhibited significant differences in expression levels or ATP binding affinities. Several kinases involved in cell cycle progression and DNA damage response quantified may be involved in the development of radioresistance in cancer cells.

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