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研究生： 陳映婷
Chen, Ying-Ting
論文名稱： 多維分離技術與標定策略用於蛋白質身分鑑定
Multidimensional Separation and Labeling Strategies for Protein Identification
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： 2009
畢業學年度： 97
語文別： 中文
論文頁數： 111
中文關鍵詞： HILIC 管柱 、蛋白質身分鑑定 、蛋白質分離
外文關鍵詞： protein separation, protein identification, Hydrophilic interaction liquid chromatography
相關次數： 點閱：98  下載：2 
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    • 生物體之蛋白質組成成分十分複雜，若要分析全部組成之蛋白質，樣品之分離步驟便非常重要。由於最終要經由質譜分析完之胜肽片段資訊做蛋白質身分鑑定，所以每次分析的胜肽樣品成分越單純，就越能得到完整之胜肽片段訊息。我的研究目標便是利用多維分離技術來將胜肽樣品的複雜度降低，以提升蛋白質身分鑑定之結果，並在蛋白質階段做分離，搭配穩定同位素二甲基標定策略來對蛋白質做身分鑑定。

    在本實驗中，運用散彈槍式蛋白質體學的方法來建立人類乳腺癌細胞MCF-7之蛋白質資料庫。利用不同之多維分離技術來分離MCF-7全細胞蛋白質溶解物，發現具有正交性之多維分離系統－ZIC-HILIC親水性管柱連接奈流C18疏水性管柱，對於蛋白質身分鑑定之結果優於C18疏水性管柱連接奈流C18疏水性管柱系統，且回收率也能大大提升。在ZIC-HILIC親水性管柱連接奈流C18疏水性管柱系統總共可以鑑定到1030個蛋白質、3059個胜肽片段；而C18疏水性管柱連接奈流C18疏水性管柱系統只鑑定到615個蛋白質、1094個胜肽片段，而兩個系統共同鑑定到的數量為蛋白質457個、胜肽片段有902個。

    在蛋白質分離部分，發現利用C18管柱分離蛋白質時，有很嚴重的樣品殘留現象，因此選擇C4管柱來分離蛋白質樣品，並且能夠用C4管柱將目標蛋白－雌激素受體純化，並用西方墨點法證明之；再搭配蛋白質標定策略－穩定同位素二甲基標定法，直接在蛋白質層面做標定，可做為研究標靶蛋白質體學的新策略。

    The sample separation is a critical step in proteome analysis due to the limitation of mass spectrometry. The more simplicity in sample composition, the more peptide information we can get. In this study, we try to reduce the sample complexity by using multidimensional separation method to obtain a good result of protein identification. Also, we separate the total proteins of human breast cancer cells (MCF-7) at protein level to purify our target protein－ estrogen receptor. We also combined ”stable isotope dimethyl labeling” to do the modification at protein level followed with trypsin digestion and mass spectrometry identification.

    In multidimensional system, we compared the number of identified proteins using two separation platforms: ZIC-HILIC-nano C18 and C18-nano C18, which only differed in first dimension. 500 μg MCF-7 whole cell lysates were digested into peptides and both systems were used for separation before mass spectrometry identification. Total of 1030 proteins with 3059 peptides were identified using ZIC-HILIC-nano C18 system and 615 proteins with 1094 peptides were identified using C18-nano C18 system. Among these results, there were 457 proteins with 902 peptides existed in both platforms. The HILIC-nano C18 system showed greater separation power since the orthogonality of two separation dimensions.

    We also compared the protein separation ability by C4 and C18 columns. We chose C4 column for separation because the mobile phase cannot elute all proteins from C18 and lots of samples retained on beads. After separating MCF-7 whole cell lysate using C4 column, all fractions were collected and ERα were detected using western blotting. The result showed that separation at protein level followed with stable isotope dimethyl labeling is a potential strategy in targeting proteomics.

    中文摘要 I 英文摘要 III 誌謝 V 表目錄 XI 圖目錄 XII 第一章 序論 1 1.1 蛋白質的基本結構 1 1.2 蛋白質體學的發展 3 1.3 蛋白質體學方法及技術 5 1.3.1 質譜技術在蛋白質體研究中的角色 6 1.3.1.1 質譜儀的構造與原理 6 1.3.1.2 串聯式質譜儀(tandem mass spectrometry，MS/MS) 14 1.3.2 蛋白質身分鑑定(protein identification) 15 1.3.2.1 高效液相層析(High-performance liquid chromatography, HPLC) 分離原理 16 1.4 實驗架構 18 第二章 利用多維液相層析結合質譜技術建立人類乳腺癌細胞(MCF-7)蛋白質資料庫 20 2.1 研究動機 20 2.2 管柱介紹 20 2.2.1 C18管柱 21 2.2.2 Hydrophilic interaction liquid chromatography (HILIC)管柱 21 2.3 人類乳腺癌細胞(MCF-7)介紹 23 2.4 材料與實驗方法 24 2.4.1 細胞培養 24 2.4.2 MCF-7全細胞萃取 24 2.4.3 蛋白質沉澱及消化 25 2.4.4 胜肽溶液之去鹽 25 2.4.5 高效能液相層析儀之分離 26 2.4.5.1 C18管柱分離 26 2.4.5.2 HILIC管柱分離 27 2.4.6 液相層析結合四極柱-飛行時間質譜儀之偵測 27 2.4.7 蛋白質資料庫鑑定 28 2.5 結果與討論 29 2.5.1 HILIC-nano C18 32 2.5.2 C18-nano C18 32 2.5.3 綜合探討 33 2.6 結論 38 第三章 利用高效液相層析儀分離人類乳腺癌細胞(MCF-7)之雌激素受體 39 3.1 研究動機 39 3.2 雌激素與雌激素受體 39 3.3 材料與實驗方法 40 3.3.1 標準蛋白質溶液之配製 40 3.3.2 MCF-7全細胞萃取 41 3.3.2.1 PBS 溶液萃取細胞法 41 3.3.2.2 RIPA 溶液萃取細胞法 41 3.3.3 高效能液相層析儀之分離 42 3.3.3.1 C18管柱之標準蛋白質分離 42 3.3.3.2 C4管柱之標準蛋白質分離 43 3.3.3.3 HILIC管柱之標準蛋白質分離 43 3.3.3.4 C4管柱之MCF-7雌激素受體純化 44 3.3.4 一維膠電泳之分離 44 3.3.5 西方點墨法 (Western blotting) 分析ERα 45 3.4 結果與討論 46 3.4.1 不同管柱對標準蛋白質之分離效果探討 46 3.4.2 C4管柱純化MCF-7之ERα 48 3.4.2.1 PBS溶液萃取結果 48 3.4.2.2 RIPA溶液萃取結果 49 3.5 綜合探討 49 3.6 結論 53 第四章 穩定同位素二甲基標定於蛋白質階段之分析 54 4.1 研究動機 54 4.2 標定策略 54 4.3 材料與實驗方法 56 4.4 數據與結果 56 第五章 總結與未來展望 61 5.1 總結 61 5.2 未來展望 61 參考文獻 63 附錄 70

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