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研究生： 古名竣
Ku, Ming-Chun
論文名稱： 雌激素化蛋白的純化與質譜偵測
Enrichment and Detection of Estrogenized Proteins Using Mass Spectrometry
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2016
畢業學年度： 104
語文別： 英文
論文頁數： 78
中文關鍵詞： 雌激素化 、蛋白富集 、可切除式生物素探針
外文關鍵詞： Estrogenization, Protein Enrichment, Cleavable Biotin probe
相關次數： 點閱：133  下載：2 
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    • 雌激素的活性代謝物”醌化兒茶酚雌激素(CE-Qs)”已被發現具有與DNA反應並主要形成脫嘌呤加合物的能力。近期一些文獻中更以放射性同位素標記法檢測結果為基礎下，推測因氧化壓力或過氧化酶而產生之醌化兒茶酚雌激素也具有於生物體內或體外與維管蛋白或微粒體蛋白產生鍵結的能力。然而能夠與兒茶酚雌激素產生結合能力的特定胺基酸種類仍然是未知的，因此我們以液相層析暨串聯式質譜儀鑑定了一系列以胺基酸作為模板檢測4-羥基雌二醇(4-OHE2)反應性的實驗，並發現到賴氨酸，半胱氨酸和組氨酸可以於4-OHE2芳環上產生共價性鍵結。此外，我們也以埃爾曼檢定法發現到4-OHE2具有與血清蛋白結合並改變其游離巰基反應性的能力。為了再更深入研究細胞內雌激素化的反應，我們設計了一個雌激素化蛋白質富集策略，其利用了雌激素相似物進行代謝標定並以一價銅催化之炔類-疊氮化物環加成反應(CuAAC)延伸探針做親和性分離。通過使用一可切除式捕獲試劑，我們成功地於體外實驗發現了與體內實驗結果具相同修飾位點的雌激素化胜肽片段，從而證明其可行性。
    這項研究揭示了一項由活性雌激素代謝物產生之潛在位點專一性蛋白質轉譯後修飾，並發現其可能涉及到雌激素代謝相關之生理紊亂。更進一步，為了鑑定並研究更多的雌激素化蛋白，我們建立了一套利用可切除式化學性探針達成蛋白純化並可應用於偵測更多內生性雌激素化蛋白質的方法。

    Catechol estrogen quinones (CE-Qs) are active metabolites of estrogen which known to react with DNA and form predominantly depurinating adducts. Several recent reports have speculated that catechol estrogen quinones produced by oxidative stress or peroxidase can also bind with tubulins or microsomal proteins in-vitro or in-vivo base on the detection of radio-isotope labeling. However, specific amino acids that can conjugate with CEs remain elusive. By investigating the reaction of 4-hydroxy estradiol (4OHE2) with a panel of amino acids using LC-MSMS, we found lysine, cysteine, and histidine can readily form covalent bonds with 4OHE2 to the aromatic A ring. Moreover, we found 4OHE2 can bind to serum proteins and alter the accessibility of the free sulfhydryl group based on the result of Ellman’s test. For a depth study of intracellular estrogenization, an estrogenized protein enrichment strategy based on E2 analog metabolic labeling and affinity isolation approach was designed. In in-vitro experiment, we successfully found estrogenized peptides which had the identical estrogenization site as that had been found in in-vivo experiment and therefore validated the feasibility of this strategy.
    This study revealed the potential formation of site-specific protein post translational modifications by active estrogen metabolites, which may be related to biological disorders associated with estrogen metabolism. Furthermore, we established an estrogenized protein enrichment strategy via a cleavable chemical probe which can be applied to identify more endogenous estrogenized proteins.

    摘要 I Abstract II Table of Contents III List of Tables IV List of Figures VI Abbreviations VIII Chapter1 : Background and Introductions 1 1.1 Brief Introduction of 4-Hydroxy 17-ß Estradiol 1 1.2 Functional Assay of Human Serum Albumin 4 1.2.1 The Antioxidant Properties 4 1.2.2 Ellman’s Test 6 1.3 Proteomics and Mass Spectrometry 7 1.3.1 Brief Introduction of Proteomics 7 1.3.2 Application of Mass Spectrometry 8 1.3.3 Bottom-Up Protein Identification by LC-MS/MS 12 1.4 Introduction of Click Chemistry 14 1.4.1 Copper(I)-Catalyzed Azide-Alkyne Cycloaddition 14 1.4.2 Cleavable Biotin Probe for Metabolic Labeling 16 Chapter2 : Experimental Methods and Materials 19 2.1 Materials 19 2.2 Amino Acid Conjugation Reactions 20 2.3 Assay of Human Serum Albumin Antioxidant Properties 22 2.4 Chemical Probe Synthesis and Feasibility Study 24 2.5 Metabolic Labeling and Enrichment In-vitro 26 2.6 Database Search 28 2.7 Conditions of Liquid Chromatography 29 2.8 Conditions of Mass Spectrometry 31 Chapter3 : Results 32 3.1 The Estrogenization of Amino Acids 32 3.2 Functional Effect of 4OHE2-adducted Human Serum Albumin 40 3.3 Chemical Probe for Estrogenized Proteins Enrichment 45 3.4 Applying Enrichment Strategy to Estrogenized Proteins In-vitro 55 Chapter4 : Conclusion 65 Appendix I 67 Appendix II 68 Appendix III 69 Appendix IV 70 Appendix V 71 Appendix VI 74 Refernce 75

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