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研究生: 魏廷宇
Wei, Ting-Yu
論文名稱: 利用質譜技術監控以及鑑定重組蛋白中間產物之雙硫鍵變化
Monitoring protein refolding process by analyzing the disulfide bonds of intermediates 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: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 145
中文關鍵詞: 固相胜肽合成蛋白質重組雙硫鍵質譜雙甲基化RADAR
英文關鍵詞: Solid phase peptide synthesis, Refolding, Disulfide Bond, Mass spectrometry, Dimethyl labeling, RADAR
DOI URL: https://doi.org/10.6345/NTNU202204353
論文種類: 學術論文
相關次數: 點閱:121下載:0
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  • 雙硫鍵是維持蛋白質活性及結構穩定性的主要轉譯修飾官能基。近幾年,固相胜肽合成技術已被廣泛應用於蛋白質藥物生產平台,但仍需透過雙硫鍵重組方能取得含正確結構及活性的產物。因此,一個能快速分析雙硫鍵的工具對於製程開發將有極大助益,也可作為蛋白質藥物必要的品管流程。生物毒素能專一的作用細胞受體,其作用機制是新型藥物開發研究的對象,本研究以固相胜肽合成之心臟蛇毒蛋白 (Cardiotoxin) 為標的,以質譜搭配RADAR軟體分析蛋白重組過程之雙硫鍵變化。藉由之雙硫鍵分析資訊,優化蛋白重組條件,純化出含正確雙硫鍵連結之產物。此外,結構分析與蛋白質活性測試結果,皆顯示重組心臟蛇毒蛋白具有與天然蛇毒蛋白質有相似β摺疊之二級結構與穿透細胞膜之活性。然而,重組過程產生之雙硫鍵錯接蛋白,則呈現不規則結構且不具活性。
    整體而言,以質譜搭配RADAR軟體可快速分析重組蛋白的雙硫鍵鍵結位點,雙硫鍵分析資訊可作為蛋白藥物的品管資料。此平台可用於中間產物的分析,藉由雙硫鍵分析資訊優化重組條件,未來可應用於製藥產業對於製造富含多半胱胺酸之生物製劑樣品。

    Disulfide bond is one of major post translation modifications for protein skeleton construction and folding maintainess. Recently, using solid phase peptide synthesis (SPPS) to generate peptide drug has been applied in pharmaceutical industry. For cysteine-rich peptides, analysis of correct disulfide bonds can be challenging. In this study, a well-developed platform using mass spectrometry was applied to profile the disulfide linkages of a synthetic peptide toxin, cardiotoxin A3 (composed by 60 amino acids with 4 disulfide bonds), during the refolding process. The bio-active cardiotoxin A3 product with correct folding was obtained by using an optimized dilution refolding method with the presence of GSSG/GSH in weak alkaline condition (phosphate buffer, pH 7.5, containing 1:3 mM ratio of GSSG/GSH). Based on the MS data, we were able to determine the protein fraction that performs exact disulfide linkage patterns as those found in native CTXA3, and optimize the manufacturing condition for peptide production. The further assays proved that the synthetic peptide product exhibited similar biochemical property and cell penetration activity as native one. The study results indicate that bioactive peptides with correct disulfide linkages could be obtained from SPPS approach with appropriate folding condition will be beneficial for the manufacturing of cysteine-rich biologics in biopharmaceutical industry. Eventually, MS-based disulfide analysis platform can provide significant information for protein structure-function study, and be the examination tool for cystein-rich bioproduct qualification.

    Abstract I 中文摘要 III 縮寫 IV 第一章 序論 1 1.1 前言 1 1.2 蛋白質雙硫鍵 1 1.3 蛋白質重組 2 1.3.1 蛋白質重組反應介紹與原理 2 1.3.2 重組反應類型 3 1.3.3 重組反應試劑與應用 3 1.3.4 蛋白質重組反應之中間產物 4 1.3.5 蛋白質重組反應路徑 5 1.3.6 蛋白質重組反應過程優化 7 1.4 蛋白質樣品介紹 8 1.4.1 蛇毒心臟毒素 8 1.4.2 人工合成蛇毒心臟毒素 8 1.5 質譜儀介紹 10 1.5.1 毛細管進樣系統(capillary inlet system) 10 1.5.2 離子源(ion source) 11 1.5.3 質量分析器 (mass analyzer) 13 1.5.4 離子偵測器 (ion detector) 14 1.6 質譜應用於蛋白質身分鑑定 15 1.6.1 胜肽碎片指紋 (peptide fragment fingerprinting, PFF) 15 1.7 蛋白質雙硫鍵鑑定 15 1.7.1 雙硫鍵鑑定方法 15 1.7.2 雙甲基化反應原理搭配RADAR (rapid assignment of disulfide linkage via a1 ion recognition)分析雙硫鍵方法 17 1.8 研究動機與目的 19 第二章 實驗材料 21 2.1 實驗樣品 21 2.2 實驗藥品 21 2.3 實驗試劑 22 2.4 實驗儀器 22 第三章 實驗方法 24 3.1 在弱酸性 (pH 6) 環境下以trypsin水解天然心臟蛇毒 24 3.2 合成心臟蛇毒重組反應使用β –Mercaptoethanol 於小體積進行重組反應 24 3.3 合成心臟蛇毒重組反應使用稀釋法搭配GSSG/GSH進行重組反應 25 3.3.1 Denature buffer (2 mL) 25 3.3.2 Refolding buffer (2 L) 25 3.3.3 實驗步驟 25 3.4 圓偏光二色光譜參數設定 27 3.5 蛋白質與胜肽活性測試 27 3.5.1 蛇毒毒性測試 27 3.6 毛細管管柱製備 28 3.6.1 製備RP-μLC column: 10 cm L × 75 μm I.D., 3 μm, 100 Å Magic C18 28 3.6.2 製備Pre-column: 2 cm L × 100 μm I.D., 5 μm, 200 Å Magic C18AQ 29 3.7 液相層析參數設定 30 3.7.1 Agilent 1100 series High Performance Liquid Chromatography in NHRI 30 3.7.2 Agilent 1100 series High Performance Liquid Chromatography in NTNU 30 3.7.3 Waters 2695 Separations Module High Performance Liquid Chromatography in NHRI 31 3.7.4 Accela 600 High Performance Liquid Chromatography in MITHRA 31 3.7.5 Waters ACQUITY Ultra Performance Liquid Chromatography in NHRI 32 3.8 質譜參數設定 32 3.8.1 Agilent MSD Trap XCT 32 3.8.2 AB Sciex QSTAR XL mass spectrometry 33 3.8.3 Waters SYNAPT G1 HDMS 33 3.9 RADAR參數設定 34 第四章 實驗結果與討論 37 4.1 天然蛇毒蛋白的雙硫鍵分析 37 4.1.1 樣品前處理 37 4.1.2 RADAR演算結果 37 4.1.3 質譜圖分析 38 4.1.4 結果與討論 38 4.2 探討以β –Mercaptoethanol作為重組反應試劑結果 39 4.2.1 實驗規劃 39 4.2.2 液相層析與質譜分子量分析 39 4.2.3 結果與討論 40 4.3 以GSSG/GSH進行心臟蛇毒重組反應 41 4.3.1 實驗規劃 41 4.3.2 液相層析串聯質譜質量偵測結果 41 4.3.3 液相層析層析譜圖結果 43 4.3.4 各重組樣品經質譜搭配RADAR演算鑑定結果 45 4.3.5 圓偏光二色光譜儀結構鑑定與生物活性測試 51 4.3.6 實驗設計討論與實驗結論 53 第五章 結論與未來展望 55 附圖 56 附表 113 參考文獻 141

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