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研究生: 張瓊文
Chang, Chiung-Wen
論文名稱: 以質譜技術評估在不同酸鹼值環境水解Avastin之雙硫鍵錯接變化
Evaluation of disulfide scrambling at various pH on Avastin digestion by 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: 2015
畢業學年度: 103
語文別: 中文
論文頁數: 93
中文關鍵詞: 雙硫鍵重組酸鹼值雙甲基化標記質譜RADAR單株抗體
英文關鍵詞: Scrambling
論文種類: 學術論文
相關次數: 點閱:111下載:0
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  •   正確的雙硫鍵摺疊影響蛋白質的活性及結構穩定性,因此對蛋白質類藥物而言,鑑定雙硫鍵的連接情況至關重要。隨著質譜技術的成熟,現今多以生化質譜方法鑑定蛋白質雙硫鍵,在此方法中需使用酵素水解蛋白質。酵素活性範圍多為弱鹼性環境,然而在弱鹼性環境下可能使蛋白質雙硫鍵還原再重新摺疊形成不同構型的雙硫鍵,造成在結構鑑定上無法判斷是樣品本身的雙硫鍵摺疊錯誤還是因實驗過程而產生的雙硫鍵重組。在本研究中,以溶菌酶(Lysozyme)及Bevacizumab(Avastin)作為樣品,控制酵素在中性以及弱酸性的溶液下進行水解,再使用雙甲基化標記結合質譜方法搭配RADAR軟體鑑定雙硫鍵,進而觀察雙硫鍵連接情形的變化,以提供最佳化鑑定雙硫鍵的實驗條件,降低雙硫鍵重組應造成的干擾。以trypsin及Lys-C在pH 6環境水解樣品,可完整鑑定到預期的含雙硫鍵胜肽,且皆未鑑定到錯接雙硫鍵。而以trypsin + Glu-C搭配變性劑rapigest在pH 6環境下水解Avastin,也可完整鑑定到預期的含雙硫鍵胜肽,且未觀察到錯接雙硫鍵。以thermolysin進行水解,不論在pH 5, 6, 7環境下都有觀察到錯接雙硫鍵的存在。本研究結果顯示,選用適當的酵素、在弱酸性的環境下進行水解反應,可有效的減少雙硫鍵重組反應的發生,雖然使用的酵素活性受到抑制,但依舊可以成功鑑定到溶菌酶及Bevacizumab中所有的雙硫鍵。

    Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure quality and functions of protein drugs. Protein digestion procedures cannot be avoided for disulfide linkage analysis in conventional manner. In order to preserve enzyme activity during protein digestion, it is commonly carried out at basic environment which increases the possibilities of disulfide bond scrambling. However; when disulfide bond rearrangement occurs, it is not quite easy to differentiate whether by sample itself or digestion process cause the scrambling disulfide linkages. In this study, optimization on digestion pH was realized for the reduction of disulfide bond rearrangement. Three sets of proteases, including trypsin plus Glu-C, thermolysin and Lys-C were used, followed by dimethyl labeling and mass spectrometry for bevacizumab (Avastin) disulfide linkage analysis. There was no scrambled disulfide bond identified at pH 6 when using Lys-C or trypsin plus Glu-C as enzymes. When thermolysin was applied, there were still scrambled disulfide bonds identified either at pH 5, pH 6 or pH 7. Nevertheless, there was fewer scrambled disulfide bonds observed at low pH. All disulfide bonds on bevacizumab can be solved with this approach. The results demonstrated that by choosing the proper enzymes, using lower digestion pH environment could reduce the degree of scrambled disulfide linkages.

    Abstract I 中文摘要 II 縮寫 III 第一章 序論 1 第一節 前言 1 第二節 蛋白質雙硫鍵 2 第三節 蛋白質藥物介紹 3 第四節 質譜儀介紹 4 第五節 蛋白質身分鑑定 7 第六節 蛋白質雙硫鍵鑑定 8 第七節 實驗原理 8 第八節 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 13 第三章 實驗方法 14 第一節 在中性及弱酸性(pH 6)環境下以trypsin水解lysozyme 14 第二節 在中性及弱酸性(pH 6)環境下以Lys-C水解Avastin 15 第三節 中性及弱酸性(pH 6)環境下以trypsin + Glu-C水解Avastin 16 第四節 以Rapigest搭配trypsin + Glu-C水解Avastin 17 第五節 在中性及弱酸性(pH 6, 5)環境以thermolysin水解Avastin 18 第六節 在pH 5環境縮短thermolysin水解Avastin時間 19 第七節 毛細管柱製備 20 第八節 層析參數設定 21 第九節 質譜參數設定 22 第十節 RADAR參數設定 24 第四章 實驗結果與討論 25 第一節 以trypsin在中性及弱酸性環境下水解lysozyme 25 第二節 以Lys-C在中性及弱酸性環境下水解Avastin 27 第三節 以trypsin + Glu-C在中性及弱酸性環境下水解Avastin 29 第四節 以thermolysin在中性及弱酸性環境下水解Avastin 31 第五章 結論與未來展望 34 參考文獻 35 附圖 40 附表 75

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