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系統識別號 U0026-0309201810254600
論文名稱(中文) 利用胜肽水平親和性純化富集策略鑑定雌激素化核蛋白
論文名稱(英文) Peptide-Level Affinity Enrichment for the Identification of Nuclear Protein Targets of Catechol Estrogens
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 106
學期 2
出版年 107
研究生(中文) 劉奕辰
研究生(英文) Yi-Chen Liu
學號 L36054338
學位類別 碩士
語文別 中文
論文頁數 80頁
口試委員 指導教授-陳淑慧
口試委員-徐睿良
口試委員-陳仲瑄
中文關鍵字 雌激素化  蛋白純化富集系統  細胞核蛋白  組蛋白 
英文關鍵字 Estrogenization  Protein enrichment  Nuclear protein  Histone 
學科別分類
中文摘要 雌激素與細胞內蛋白質共價結合會導致功能失常,若其代謝物與 DNA 鍵結成加成物更會提高罹癌的風險,因此研究雌激素化蛋白質是刻不容緩的!近年研究發現,雌激素於人類胰島素上的修飾會影響其訊號傳輸、於人類血清白蛋白上的修飾會影響其抗氧化及運輸物質的功能。雌激素化修飾的重要性已明確可知,但礙於其修飾微小,因此就算以靈敏度極高的質譜儀也難以偵測到真實樣品中所有的雌激素化蛋白,特別是低含量的蛋白,因此需要純化策略,排除真實樣品中的干擾物質與基質,只針對雌激素化的蛋白做鑑定。
在此,實驗室開發出一套純化富集系統,先利用鍵擊化學-疊氮基與炔類環化加成反應,穩定得結合含有雙硫鍵裂解點的生物素化學探針與雌激素代謝物,搭配目前已知最強非共價鍵鍵結-生物素與鏈親合素的親和力,將非雌激素的蛋白清洗掉且去除真實樣品的干擾物質,進而達到純化富集的效果。最後再搭配雙甲基標定法以 D/H 比值判斷其可信度,已成功從肝臟組織中鑑定出 374 個極可能被雌激素修飾的蛋白,其中有許多影響細胞內氧化還原能力的酵素。
然而,還有許多非雌激素化的蛋白與干擾物質尚未去除,因此衍伸出了改良的純化富集系統。原先的純化策略在鏈親合素鍵結步驟後,接著切斷生物素探針上的雙硫鍵裂解點,使抓住的雌激素化蛋白沖堤收集起來並接著做酵素水解。然而得到的產物也會包含許多非雌激素鍵結的胜肽片段,因此新的純化策略在鏈親合素鍵結步驟後,接著使用兩種酵素做水解,並去除大多數非雌激素化修飾的胜肽片段,再將雌激素化胜肽沖堤下來,達到更好的富集效果,提供了更多雌激素化修飾的片段,且提高可信度與一次質譜的訊號強度及清晰的二次質譜圖。
將改良的純化系統,應用在細胞核蛋白的純化,鑑定到了108個蛋白,含有73個蛋白有雌激素修飾,有7個極可信的雌激素修飾核蛋白。其中含有組蛋白甲基轉移酶(Histone methyltransferase),此酵素會影響組蛋白上甲基化修飾的程度,影響片段基因的表現與否。可望對轉譯後修飾與組蛋白上的雌激素位點,有更加深入的了解,藉此提供生物醫學科學家更多訊息。
英文摘要 Catechol estrogens (CEs) are known to react with DNA and form adducts that are believed to be the initiators of estrogen-induced tumorigenesis. CEs covalently interaction with cellular proteins, referred as estrogenized proteins, influencing molecular function. Their conjugation sites are Cysteine, Lysine and Histidine. There is no feasible method to identify their binders in a living system. Here, we developed a click chemistry-based affinity enrichment to identify protein targets of CEs using liquid chromatography-mass spectrometry (LC-MS).
Human insulin was estrogenized by co-incubated with 4-hydroxyl ethinyl estradiol (4OHEE2) and used as model protein to optimize the click reaction yield. Human serum albumin (HSA) was estrogenized to optimize the enrichment efficiency by using digested peptide molecules. Via affinity enrichment, we can remove most of unmodified proteins, but still have many unmodified peptides with high intensity that influence detection and identification. In new strategy, we did enzyme digest after removing unmodified proteins then remove unmodified peptide again with more volumes buffer wash. CEs conjugated peptides then elute and detect by LC-MS/MS.
New strategy provides more CE-modified peptide with higher intensity and clearer MS2 spectra. With dimethyl labeling can achieve efficient of enrichments results. We also extract nuclear proteins by 4OHEE2 treatment and detect Histone family with CE-modified peptides that provide information of post-translation modification related with breast cancer. This method can be widely applied to study cellular targets of CEs in any biological systems. Thus, we expect the peptide-level enrichment strategy will be useful to explore more estrogenized proteins and modified site information.
論文目次 中文摘要 I
Extend Abstract II
致謝 VII
目錄 VIII
表目錄 XI
圖目錄 XII
簡寫表 XV
第一章 緒論 1
第二章 背景介紹 2
2.1 雌激素與雌激素化蛋白簡介 2
2.2 蛋白質體學與質譜應用 5
2.2.1 蛋白質體學簡介 5
2.2.2 質譜技術於蛋白質鑑定應用 6
2.2.3 常見質譜技術 7
2.3 目標蛋白簡介 11
2.3.1 人類胰島素 11
2.3.2 人類血清白蛋白 14
2.3.3 細胞組蛋白 15
2.4 純化策略介紹 18
2.4.1鍵擊化學(Click chemistry)-疊氮基與炔類環化加成 18
2.4.2 代謝標定(Metabolic Labeling)及生物素鍵結 20
2.4.3 雙甲基標定法(Dimethyl labeling) 22
第三章 實驗方法 23
3.1 實驗藥品與儀器 23
3.1.1 實驗藥品 23
3.1.2實驗儀器與器材 24
3.2 生物素化學探針的合成與純化 25
3.3 細胞培養及蛋白萃取 26
3.3.1 細胞培養條件、繼代、凍存及恢復 26
3.3.2 雌激素處理及蛋白質萃取 28
3.4 雌激素化蛋白純化與標定過程 31
3.4.1 人類胰島素 31
3.4.2人類血清白蛋白 33
3.4.3 細胞核蛋白 35
3.5 層析參數設定與質譜設定 37
3.6 資料庫搜尋軟體設定 38
第四章 結果與討論 40
4.1 鍵擊化學步驟優化 40
4.1.1 參數調整 40
4.1.1.1 銅離子濃度 41
4.1.1.2 化學探針濃度與樣品比例 42
4.1.1.3 配位基濃度 43
4.1.1.4 還原劑濃度 44
4.1.1.5 反應時間 45
4.1.1.6 反應溫度 47
4.1.2 純化效果確立 49
4.2 細胞蛋白萃取 50
4.2.1 細胞質蛋白 50
4.2.2 細胞核蛋白 57
4.2.3 組蛋白 60
4.3 新舊純化策略效果比較 61
4.3.1 人類胰島素蛋白 61
4.3.2 人類血清白蛋白 63
4.4 雌激素化細胞核蛋白純化 67
4.4.1 細胞核被雌激素修飾位點 67
第五章 結論 73
第六章 參考文獻 74
附錄 79
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