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研究生:林玄聰
研究生(外文):Hsuan-Chung Lin
論文名稱:毫秒氫氘交換技術(millisecondH/Dexchange)結合電噴灑游離質譜法(ESI/MS)研究蛋白質的三度立體結構
論文名稱(外文):Millisecond H/D Exchange Combined with Electrospray Ionization Mass Spectrometry to Study Protein’s Structure
指導教授:謝建台
指導教授(外文):Jentaie Shiea
學位類別:碩士
校院名稱:國立中山大學
系所名稱:化學系研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:109
中文關鍵詞:串聯質譜蛋白質結構融合微滴電噴灑游離質譜法氫氘交換胜肽鍵上的氫電噴灑游離法
外文關鍵詞:FD-ESI/MSamide hydrogenESIProtein structureMS/MSH/D exchange
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融合微滴電噴灑游離質譜法(Fused Droplet / Electrospray Ionization Mass Spectrometry, FD-ESI/MS)是近年來由本實驗室所發展出來的一種質譜游離技術,此技術與傳統電噴灑游離法(Electrospray Ionization, ESI)不同,傳統ESI法是藉由施加一高電壓於低流速的樣品溶液,直接使樣品溶液進行電噴灑游離;而FD-ESI則是採兩步驟來進行游離:樣品溶液先以霧化器將其霧化成微小液滴,再導入一以酸性甲醇液進行傳統ESI的區域,樣品液滴在和帶電荷甲醇液滴進行融合後,再由所形成的新融合液滴進行電噴灑,以產生具多價電荷的分析物離子。

氫氘交換(Hydrogen/Deuterium Exchange, H/D exchange)是研究蛋白質的三度空間結構方法之一,其原理是利用蛋白質結構中含氫(hydrogen)的位置,以含有氘(deuterium)的溶液環境下,使得蛋白質分子上的氫被氘取代而進行H/D交換,一般發生氫氘交換的位置則是在NH, NH2, OH, COOH, SH等官能基上的氫。當蛋白質分子結構上其中一個氫被氘取代之後,其分子量將會增加1 Da,因此以質譜檢測即可看出完成H/D交換後的質量差異,進而提供蛋白質分子構形變化情形的訊息。

本實驗則是依據FD-ESI特有的性質與H/D交換技術結合而發展出millisecond H/D exchange(毫秒氫氘交換)技術,用以彌補傳統H/D交換研究蛋白質結構技術的缺失。研究中將此技術應用於探討胺基酸(amino acid)、胜肽(peptide)等分子內可快速進行 H/D 交換的氫數目,再以串聯質譜(MS/MS)法來證明主要的交換位置;此技術最大優點是可以在極短時間內(約millisecond)快速看到蛋白質分子構形變化的過程,亦可區分出不同種類來源且具微小序列差異的蛋白質(如牛、狗、馬之色素細胞c所造成立體結構的差異),對於具不同數量的雙硫鍵(disulfide bond)蛋白質,也可依其在毫秒時間內迅速進行H/D交換的數目不同而作區分,因此此技術可以應用在探討蛋白質分子三度空間結構訊息與變化。
目 錄

論文摘要………………………………………………………………….i
目錄……………………………………………………………………...iv
圖目錄…………………………………………………………………..vii
表目錄…………………………………………………………………...xi
壹、緒論………………………………………………………………….1
一、前言…………………………………………………………….1
二、融合微滴電噴灑游離質譜法發展與原理簡介……………….4
2.1.電噴灑游離質譜法發展歷史與原理……………………..4
2.1.1.電噴灑游離質譜法發展歷史……………………….4
2.1.2.電噴灑游離質譜法的原理及機制………………….5
2.2.融合微滴電噴灑游離質譜法的發展與原理..…………..10
2.2.1.融合微滴電噴灑游離質譜法發展歷史…………...10
2.2.2.融合微滴電噴灑游離質譜法原理...………………12
2.2.3.串聯質譜(Tandem Mass Spectrometry或MS/MS)法原理……………………………………………..14
三、電噴灑游離質譜法結合氫氘交換在蛋白質結構的研究……18
3.1.蛋白質的組成與結構…………………………………....18
3.2.影響蛋白質構形的因素………………………………....25
3.3.電噴灑游離質譜法於蛋白質結構的研究……………....27
四、氫氘交換技術發展歷史與原理……………………………..32
五、論文目標……………………………………………………..38
貳、實驗………………………………………………………………..41
一、儀器裝置……………………………………………………..41
1.質譜儀……………………………………………………...41
2.超音波霧化器………….…………………………………..42
3.微量注射幫浦………………….…………………………..43
二、實驗試劑與配置方法………………………………………..44
三、實驗方法……………………………………………………..47
1.傳統H/D交換部分
分析物直接配置在氘溶液中進行電噴灑...……………...47
2.毫秒H/D交換部分
分析物配置在水溶液中進行毫秒氫氘交換……………..48
參、結果與討論…………………………………………………………49
一、 毫秒氫氘交換技術應用在有機分子與胺基酸分子之研究……………………………………………………………49
1. 以1,12-Diaminododecane進行氫氘交換實驗………......49
2. 以reserpine進行氫氘交換實驗……………………….....54
3. 以arginine進行氫氘交換實驗…………………………..58
二、毫秒氫氘交換技術應用在胜肽分子之研究………………..62
1. 以Pro-Leu-Gly amide進行氫氘交換實驗……….....…...62
2. 以leucine-enkephalin進行氫氘交換實驗.………………67
3. 以bradykinin進行氫氘交換實驗..………………………75
4. 以angiotensin I進行氫氘交換實驗..…………………….80
二、毫秒氫氘交換技術應用在蛋白質分子之研究………………85
1.以同種類但不同來源的細胞色素c進行氫氘交換實驗…85
2.以蛋白質結構上的雙硫鍵多寡來進行氫氘交換實驗……91
肆、結論…………………………………………………………………99
伍、參考文獻……………………………………………………….….102
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