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研究生: 葉沛柔
Yeh, Pei-Jou
論文名稱: 利用質譜技術鑑定甲型流感病毒血球凝集素H7蛋白的雙硫鍵鍵結
Characterization of Disulfide Linkages for Influenza A Virus Hemagglutinin H7 Protein Using Mass Spectrometry
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 105
中文關鍵詞: 甲型流感病毒H7N9亞型血球凝集素雙硫鍵雙甲基化質譜RADAR
英文關鍵詞: H7N9, hemagglutinin, Disulfide bond, Dimethyl labeling, Mass spectrometry, RADAR
DOI URL: http://doi.org/10.6345/NTNU201900359
論文種類: 學術論文
相關次數: 點閱:113下載:0
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  • 雙硫鍵為影響蛋白質功能與活性的關鍵結構因子,對於可作為疫苗的重組抗原蛋白來說,雙硫鍵會影響其摺疊以及結構,錯接的雙硫鍵會嚴重的影響抗原與抗體結合的特異性,因此蛋白質正確雙硫鍵鍵結的鑑定是極重要的。本篇研究中,我們利用雙甲基化反應搭配液相層析質譜技術以及RADAR軟體的輔助,鑑定甲型流感病毒H7N9病毒顆粒以及其重組的血球凝集素H7蛋白: H7蛋白三聚體、H7蛋白單體以及Dsbc-H7、Dsbc-HA1。蛋白質樣品利用不同的酵素組合在酸性環境下水解,在本研究中使用包含: 胰蛋白酶 (trypsin)、trypsin + chymotrypsin、trypsin + asp-n等組合。使用trypsin + asp-n的酵素組合可在全部的樣品中鑑定到四個雙硫鍵鍵結,此為鑑定H7蛋白最佳的酵素組合。雙硫鍵片段C272-C296可在H7N9樣品中被鑑定到,而雙硫鍵片段C4-C458則在H7-trimer中被鑑定到。此雙硫鍵分析方法可對於蛋白質活性提供重要的資訊,據我們所知,此篇為首次利用雙硫鍵分析的方法探討甲型流感病毒H7N9血球凝集素H7重組蛋白活性的研究。

    Disulfide linkage is a crucial structure factor that affect the functionality and activity of proteins. As to the recombinant antigen proteins used for vaccine production, disulfide bonds affect their folding and structure. Disulfide bond scrambling would greatly influence the specificity of antigen-antibody binding. Thus, confirmation of the correct disulfide bond pairings is important. In this study, dimethyl labeling coupled with LC-MS/MS and RADAR algorithm was used to identify the disulfide bonds in influenza A virus particle H7N9 and its recombinant H7 proteins, including H7-trimer, H7-monomer, Dsbc-H7, and Dsbc-HA1. They were digested with different protease combinations including trypsin, trypsin/chymotrypsin, and asp-n in acidic condition. Four disulfide bonds were identified in all samples using trypsin plus asp-n, which gave the most disulfide bond identifications among all protease combinations. The disulfide linkage peptides, C272-C296 and C4-C458, can be identified only in H7N9 virus particle and H7-trimer, respectively. Eventually, this proposed disulfide linkage analysis can provide significant knowledge for protein activity study. To our knowledge, this is the first report using LC-MS/MS for the analysis of disulfide linkages on influenza A virus hemagglutinin H7 protein.

    謝誌 I 中文摘要 II Abstract III 目錄 IV 圖目錄 VII 表目錄 XII 第一章 緒論 1 第一節 前言 1 第二節 蛋白質樣品介紹 2 第三節 蛋白質雙硫鍵 4 第四節 蛋白質雙硫鍵鑑定方法 5 第五節 質譜儀介紹 6 (一) 質譜儀技術 6 (二) 串聯質譜分析 7 第六節 蛋白質身份鑑定與雙硫鍵鑑定 8 (一) 蛋白質身分鑑定 8 (二) 以雙甲基化反應搭配RADAR分析雙硫鍵 9 第七節 研究動機與目的 11 第二章 實驗材料 12 第一節 實驗樣品 12 第二節 實驗藥品 12 第三節 實驗試劑 12 第四節 實驗儀器 12 第三章 實驗方法 14 第一節 在弱酸性 (pH 6) 環境下以trypsin 水解 H7N9 14 第二節 在中性及弱酸性 (pH 6) 環境下以 trypsin + chymotrypsin 水解 H7N9, Dsbc-H7, H7-monomer, H7-trimer 14 第三節 在弱酸性 (pH 6)環境下以 trypsin + asp-n 水解 H7N9, Dsbc-H7, Dsbc-HA1, H7-monomer, H7-trimer 16 第四節 液相層析參數設定 17 第五節 質譜參數設定 18 第六節 RADAR 參數設定 18 第四章 實驗結果與討論 20 第一節 以trypsin在弱酸性環境下水解H7N9 21 (一) 實驗規劃 21 (二) RADAR搜尋結果 21 (三) MS/MS圖譜比對 24 (四) 結果與討論 25 第二節 以 trypsin + chymotrypsin 在中性及弱酸性環境下水解 H7N9, Dsbc-H7, H7-monomer, H7-trimer 28 (一) 實驗規劃 28 (二) RADAR搜尋結果 31 (三) MS/MS圖譜比對 31 (四) 結果與討論 31 第三節 以 trypsin + asp-n 在弱酸性環境下水解 H7N9, Dsbc-H7, H7-monomer, H7-trimer 35 (一) 實驗規劃 35 (二) RADAR搜尋結果 38 (三) MS/MS圖譜比對 38 (四) 結果與討論 38 第五章 結論與未來展望 43 附圖 45 參考文獻 103

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