同位素化學標記法搭配質譜技術進行發炎反應動物模式之差異蛋白質體學研究

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2015

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Proteomics is a large-scale comprehensive study of a specific proteome, including information on the levels of different types of proteins, their modifications and variations, as well as their interactions and networks, in order to understand biological processes. Recent successes clearly show that mass spectrometry-based proteomics as an essential tool for molecular and cellular biology and for the rising field of systems biology. Two-dimensional fractionation is a useful tool to increase proteome coverage and the dynamic range than single-dimensional LC. In part I of this dissertation, various peptide fractionation strategies that are used for 2D (two-dimensional) separations were evaluated. The use of SCX x RPLC for desalted samples provided superior results in protein identification. These approaches are complementary and allowed 43% more peptides to be identified, when compared with a single fractionation strategy. In part II, LTQ-PQD parameters were optimized in order to used isobaric tags technology for quantitative proteomics. The number of microscans and the target value are the most critical factors in producing intense reporter ions for quantitation. The appropriate normalized collisional energy range for PQD could be very narrow and must be carefully determined. The optimized LTQ-PQD parameters were introduced to a murine air pouch membrane in part III. iTRAQ-based approach coupled with offline 2D LC-MS/MS proteomics technology was applied to analyze the protein expression profile using an inflamed murine air pouch membrane as a model. Statistical analyses revealed that 317 proteins are differentially expressed, at least at one time point, after the MSU treatment, that they are mainly involved in the complement system and activation of NALP3 inflammasome. Moreover, the TCA cycle was found to be down-regulated at both the translational and transcriptional levels. Lastly, pyruvate carboxylation was found to be a potential target for an anti-gout treatment. These results provide novel insights into the nature of gouty inflammation.

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二維分離, 液相層析, 質譜, 差異蛋白質體學, 痛風, Two-dimensional separation, LC-MS/MS, Pulsed-Q dissociation, Differential proteomics, Monosodium urate crystal, Gout

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