液相層析質譜已成為主要研究蛋白質體學和代謝的工具。本論文中，液相層析質譜應用於進行三個蛋白質體學和代謝研究：一、大鼠肝微粒體酵素及其亞型的性別-相關差異表現（第二章）。 二、定量分析乳癌細胞之內源性雌激素及其代謝產物（第三章）。 三、兒茶酚雌激素代謝物修飾蛋白質（第四章）。這些研究為新的假說在雌激素作用上為帶來線索。
在第二章中，我們採用散彈槍定量蛋白質體學方法加上穩定同位素二甲基標定，二維逆相勝肽的分離和串聯質譜（MS）探討大鼠肝微粒體蛋白之性別差異表現。結果總共391個蛋白質被鑑定與定量，其中56％的蛋白質是酵素。我們確定了53個亞型，其中包括21個 CYP450的亞型。經由定量和統計評估，我們歸類出28酵素為公的在數量上佔優勢、21酵素為母的在數量上佔優勢，以及125性別無關的酵素。進一步以質譜分析雄性和雌性大鼠之催化速率，可證實雄性和雌性大鼠在CYP2D1的表現上有約30%的差異。
在第三章中，我們報導了定量液相層析串聯質譜法，經由簡單的乙酸乙酯萃取和丹磺酰氯衍生化後，利用選擇性反應監測分析MCF - 7乳癌細胞之雌激素和雌激素代謝物在細胞外和細胞內的含量。結果顯示，未經處理的條件下，微量的雌酮和雌二醇（<0.9 x10-15克/細胞）同時存在於細胞外和細胞內，但二甲基亞碸可誘導細胞產生微量的雌酮和雌二醇（<2.25 x10-15克/細胞）。經雌二醇處理，不僅大大增加雌酮和雌二醇在細胞內的量（60 x10-15克/細胞）和細胞外的量（3000 x10-15克/細胞），細胞外代謝物的量也大幅增加（0.6至25x10-15克/細胞）。這些數據暗示，MCF - 7乳腺癌細胞以雌二醇處理24小時後，雌激素代謝物可以迅速生成並排到細胞外，而二甲基亞碸溶劑可能引起輕微的雌激素作用。
在第四章中，我們進行了質譜為基礎的研究，系統地探討兒茶酚雌激素與氨基酸、勝肽和蛋白質在生物條件下的反應。我們稱此類蛋白被修飾上兒茶酚雌激素現象為蛋白質雌激素化。我們發現，活化的兒茶酚雌激素可以很容易與含有親核性官能基之氨基酸 (例如離氨酸，組氨酸和半胱氨酸)進行Michael addition。利用一次質譜和二次質譜在CID解離或ETD解離（ETD）條件下，我們確認，活化的兒茶酚雌激素與在勝肽上的離氨酸，組氨酸和半胱氨酸殘基反應，但其他殘基則未被發現。我們也發現了蛋白質可在生物緩衝條件下被雌激素化。而且確定生物樣品中蛋白質雌激素化的研究將持續進行。

Liquid chromatography-mass spectrometry (LC-MS) has become the major tool for proteomic and metabolic studies. In this thesis, LC-MS was applied to conduct three proteomic and metabolic studies: 1. Gender-dependent differential expression of rat microsomal enzymes and their isoforms. (Chapter two) 2. Quantitative analysis of endogenous estrogens and their metabolites in breast cancer cells. (Chapter three) 3. Protein modification by catechol estrogen metabolites. (Chapter four) These investigations shed lights into novel hypotheses for estrogen action.
In chapter two, we applied quantitative shotgun proteomics approach coupled with stable isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. We identified 53 isoforms including 21 isoforms of CYP450s. By quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes and 21 female dominant enzymes; and 125 sex independent enzymes. The catalyzing rate of CYP2D1 was further proved to exhibit significant differences between male and female rats by MS analyses.
In chapter three, we reported a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing selective reaction monitoring (SRM) to analyze estrogens and estrogen metabolites in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Results indicate that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce formation of E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6 to 25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24 hour of treatment and DMSO solvent could potentially induce slight estrogen effects.
In chapter four, we conducted a MS-based study to systematically investigate the reactivity of catechol estrogens towards aminoacids, peptides, and proteins under biological conditions. We term such protein modifications by catechol estrogens as protein estrogenylation. We found the activated catechol estrogens could readily react with amino acids of lysine, histidine, and cysteine which contain nucleophilic functional groups via Michael addition. Using MS and MSMS under collision induced dissociation (CID) and electron transfer dissociation (ETD), we confirmed that the activated 4-OHE2 reacts with lysine, histidine, and cysteine residues on a synthetic peptide but not on other residues. We had also detected protein estrogenylation under biological buffer conditions. Studies to identify protein estrogenylation in biological samples will be conducted in the future.

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