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研究生： 林峰賢
Lin, Fong-sian
論文名稱： 利用質譜分析半胱胺酸胜(月太)結合螢光標籤親和層析法在蛋白質體學之研究
Fluorescein Affinity Enrichment of Cysteine-containing Peptides for MS-based Proteomics
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： 2007
畢業學年度： 95
語文別： 中文
論文頁數： 94
中文關鍵詞： 螢光親和層析法 、人類乳腺癌細胞 、同位素親和標籤 、質譜
外文關鍵詞： Mass spectrometry, ICAT, fluorescein affinity chromatography, MCF-7
相關次數： 點閱：166  下載：1 
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    • 存在於生物體中的蛋白質成千上萬種，於不同細胞、組織、器官中的蛋白質也有可能產生不同的後轉譯修飾，造就了生物體中蛋白質組成的複雜性，因此分析微量且具可能致病的蛋白，變成了研究者極力想要去瞭解的目標。排除高含量蛋白質的訊號干擾，顯示出具有研究性的微量蛋白，使得可以利用質譜進行研究。
    本篇論文採用類似於同位素親和標籤（isotope coded affinity tag, ICAT）的策略，使用具有鍵結於半胱氨酸的特異性基團，再利用此基團達到半胱氨酸胜肽的純化。藉由此策略達到減少樣品複雜度，以及偵測微量蛋白質的目標。
    實驗中使用5-(Iodoacetamido)fluorescein（5-IAF）鍵結於蛋白質的半胱氨酸上，再將酵素消化產生的胜肽混和物注入填充有Poros 20 MC的層析管柱中，由於5-IAF會與粒子產成親和作用，藉此達到純化半胱氨酸胜肽，再使用質譜進行分析。所使用的反應試劑及粒子相較於ICAT更為便宜且操作容易，具有相同的減少樣品複雜度及微量蛋白偵測效果。藉由此純化的結果，再搭配雙甲基標定（dimethyl labeling）達到定量的效果，便完成了整個ICAT策略。
    實驗中，藉由ovalbumin及bovine serum albumin兩種標準品建構出整個實驗流程。MALDI MS的偵測中，證明確實有純化出螢光標籤標記的半胱氨酸胜肽；再將樣品送入LC / Q-TOF tandem MS進行MS/MS分析，可以在MASCOT的比對下，於蛋白質資料庫中找出正確的蛋白質。而且螢光標籤標記的半胱氨酸胜肽在MS/MS圖譜上，發現具有來自鍵結5-IAF的胜肽所產生的報導離子（reporter ion），藉由此報導離子的產生，可以於LC / Q-TOF tandem MS使用precursor ion discovery的質譜偵測模式，針對具有螢光標籤標記的半胱氨酸胜肽分析，排除非半胱氨酸胜肽的偵測，提高半胱氨酸胜肽的偵測效率。
    最後，將此螢光親和層析法（fluorescein affinity chromatography）套用至分析MCF-7細胞株上，可以在MALDI MS發現有特定質量之胜肽訊號於最後被沖提出；在LC-ESI-Q/TOF上發現採用此策略可以找到數種蛋白質，而這些蛋白質經由一般樣品處理步驟是無法取得的，證明此策略確實具有偵測微量蛋白的效果。

    Mass spectrometric analysis of proteomics is critically dependent on optimal sample purification. In this study, we demonstrate a novel approach to selectively enrich the fluorescein-labeled proteins prior to mass spectrometric analysis, named fluorescein affinity chromatography (FAC). 5-(Iodoacetamido)fluorescein can specifically react to the cysteine in peptides without any byproducts. Efficiency of the IAF-labeled procedure is nearly complete and no observable byproducts were found in LC-MS/MS analysis. The fluorescein derivatives were shown high adsorption toward some porous metal-chelating material such as Poros MC 20 beads. IAF-labeled proteins or peptides could bind the porous material tightly under acidic condition and meanwhile, non-labeled species were washed out with 0.1% TFA. The dye-labeled moiety, therefore were concentrated and eluted by slightly alkaline buffer and detected directly by the naked eye.
    The tryptic digest of IAF-modified ovalbumin and BSA were used as model proteins to demonstrate the efficiency of this approach and the result was examined by mass spectrometry. MALDI spectra indicate the trapping efficiency for cysteine-containing phosphopeptides of ovalbumin was complete and it lead to a great reduction in sample complexity. Several peptides containing adjacent cysteine residues were found to be labeled completely without byproducts. These results indicated that this method is practical in extraction of cysteine-containing peptides. Moreover, a CID fragment of m/z 422 is generated from the IAF-labelled cysteine-containing peptides and it can be conveniently used as the reporter ions. Combining the enrichment method described in this study and precursor ion scan, we were able to increase the percentage of cysteine-containing peptides identified from MS analysis. By applying the proposed method to a complicated sample such as MCF-7 cell lysate, we were able to identify low abundant cysteine- containing proteins such as Alpha-2-HS-glycoprotein precursor, Glia maturation factor beta, Wiskott-Aldrich syndrome protein family member 4, Acyl carrier protein, mitochondrial precursor and Transportin-2.
    Overall, this method reduced the complexity of peptide mixtures by enriching cysteine-containing peptides. The method is simple and can be traced by the color of dyes. Moreover, the unique of fragment m/z 422 associted with the labeling provides a convenient means for MS-based signal enrichment by precursor ion scan. There are potential uses of the method such as cysteine mapping of proteins or protein profiling by coupling the method with stable isotope labeling.

    目 錄 中文摘要 I 英文摘要 III 誌謝 V 目錄 VI 表目錄 X 圖目錄 XI 附錄 XIII 第一章 序論 1.1 前言 1 1.2 蛋白質學的發展 2 1.2.1 何謂蛋白質體學 2 1.2.2 蛋白質體的進展 4 1.2.3 蛋白質的基本組成 5 1.3 質譜技術用於蛋白質的偵測 8 1.3.1 基質輔助雷射脫附游離法（MALDI） 9 1.3.2 電噴灑游離法（ESI） 11 1.4 生物醫學結合蛋白質體於質譜上的應用 12 1.4.1 質譜技術結合蛋白質體學用於癌症標靶蛋白質的發現 13 1.4.2 質譜技術結合蛋白質體學用於新藥的開發 15 1.5 實驗架構 15 第二章 螢光標籤親和色層法（fluorescein affinity chromato- graphy, FAC）之開發 2.1 實驗材料 19 2.1.1 標準蛋白樣品 19 2.1.2 實驗試劑及酵素 19 2.1.3 實驗材料及儀器設備 20 2.2 樣品及溶劑配製 22 2.3 實驗方法及流程 23 2.3.1 蛋白質鍵結染料 23 2.3.2 樣品純化 24 2.3.3 蛋白質的消化 25 2.3.4 螢光標籤親和色層法之操作 25 2.3.4.1 螢光標籤親和色層管柱及系統架設 25 2.3.4.2 螢光標籤標記之半胱氨酸胜肽的純化 27 2.3.5 MCF-7細胞內蛋白之萃取 28 2.4 MCF-7蛋白質資料庫之建立 29 2.4.1 solution phase IEF蛋白質分離 29 2.4.2 Strong anion exchange蛋白質分離 30 2.5 基質輔助雷射脫附質譜儀對螢光標籤標記半胱氨酸胜肽之偵測 30 2.5.1 儀器裝置 30 2.5.2 MALDI MS樣品製備及偵測 32 2.6 液相層析結合四極柱-飛行時間質譜儀之偵測 33 2.6.1 儀器裝置 33 2.6.2 LC- MS樣品製備及偵測 37 2.7 蛋白質之資料庫與鑑定方式 37 第三章 實驗結果與討論 3.1 螢光標籤反應完整性之探討 44 3.2 MALDI MS與LC/Q-TOF tandem MS靈敏度之比較 48 3.3 螢光標籤標記半胱氨酸胜肽之純化 50 3.4 螢光標籤標記的半胱氨酸胜肽於MS/MS分析 54 3.5 螢光標籤標記之半胱氨酸胜肽的PID（Precursor ion discovery） 56 3.6 真實樣品結合螢光標籤親和標籤層析法之探討 61 3.6.1 真實樣品於MALDI MS偵測之結果 62 3.6.2 真實樣品於LC-ESI-Q/TOF偵測之結果 64 第四章 人類乳腺癌細胞（MCF-7）蛋白質資料庫 4.1 細胞株簡介 69 4.2 MCF-7蛋白質資料庫 69 4.2.1 sIEF fraction 1 (pH 3.0~4.6) 70 4.2.2 sIEF fraction 2 (pH 4.6~5.4) 71 4.2.3 sIEF fraction 3 (pH 5.4~6.2) 74 4.2.4 sIEF fraction 4 (pH 6.2~7.0) 76 4.2.5 sIEF fraction 5 (pH 7.0~10.0) 79 第五章 總結與未來展望 5.1 總結 80 5.2 未來展望 81 參考文獻 83

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