簡易檢索 / 詳目顯示

研究生: 謝育廷
論文名稱: 利用雙甲基化搭配液相層析串聯式質譜技術進行蛋白質藥物Trastuzumab之雙硫鍵結鑑定分析
Disulfide Linkage Analysis of Trastuzumab Using Dimethyl Labeling and Liquid Chromatography Tandem Mass Spectrometry
指導教授: 陳頌方
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2011
畢業學年度: 99
語文別: 中文
論文頁數: 92
中文關鍵詞: 單株抗體雙硫鍵a1離子蛋白質藥物
英文關鍵詞: Monoclonal antibody, Disulfide bond, a1 ion, Protein pharmaceutical
論文種類: 學術論文
相關次數: 點閱:153下載:14
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 雙硫鍵在穩定蛋白質的三級結構和維持其生化活性上扮演重要的角色,同時,雙硫鍵的完整鍵結與否,常常關係到蛋白質藥物的品質.傳統上利用質譜分析蛋白質雙硫鍵的方法,常採用間接鑑定方式,比較經過還原前和還原以後含有cysteine胜肽質譜圖的差異,雙硫鍵的兩個胺基酸cysteine經還原後會接上兩個氫,而使該胜肽鏈的分子量多2 Da,但當同一個蛋白質裡含有複雜且多條的雙硫鍵時,就會產生更多樣的排列組合,所以這樣的方法,並不適用於分析具有多條複雜雙硫鍵的蛋白質.
    在本篇研究中,我們提出一套鑑定雙硫鍵的方法,應用於蛋白質藥物trastuzumab上.在非還原的條件下,利用雙甲基化胜肽在Q/TOF質譜中,CID碎片會有a1離子訊號被提升的現象,搭配計算軟體RADAR掃描a1離子及其對應分子量,並與資料庫中含有cysteine的胜肽組合去比對,藉此鑑定出雙硫鍵的位置.此外,將針對不同的蛋白酶搭配,多種蛋白質水解與雙甲基化的最佳條件作測試,以期可以在最短的時間內,鑑定到最多正確的雙硫鍵數目.最後,在最佳化實驗條件下,利用RADAR對a1離子作自動化的掃描,成功的鑑定出trastuzumab上七條雙硫鍵位置.
    本篇研究提供一套快速且正確鑑定雙硫鍵的方法,適用於例行的蛋白質藥物結構的鑑定,可以發展成一套完整有效的蛋白藥物品管方式.

    Disulfide bond formation plays a critical role in stabilizing protein tertiary structure thus maintaining its biological activity. It also infers the quality of recombinant protein pharmaceuticals. In this study, a simple and rapid method was demonstrated for disulfide bond identification of an IgG drug trastuzumab.
    Dimethyl labeling was applied to proteolytic peptides in nonreduced condition, which exhibit enhanced a1 ion signals in CID; multiple a1 ions can be observed due to multiple N-termini. Computational software RADAR was also developed to perform the automatic a1 ion screening followed by searching for molecular weight match among the cysteine-containing peptides. Selections of appropriate proteases combination and buffer pH for dimethyl labeling were investigated. The results (peak lists) were dealt with custom-made software RADAR to screen a1 ions, and found the corresponding molecular weight for disulfide bond assignment.
    Seven unique disulfide linkages of trastuzumab were successfully identified using automatic a1 ion screening and RADAR with optimized experimental conditions. This approach provides accurate and efficient solution, and it has great potential for routine structural characterization of protein pharmaceuticals.

    圖目錄..................................................................Ⅲ 表目錄..................................................................Ⅴ 中文摘要................................................................Ⅵ Abstract................................................................Ⅶ 縮寫....................................................................Ⅷ 第一章 導論..............................................................1 第一節 蛋白質結構.................................................1 第二節 生物藥品...................................................3 第三節 蛋白質的結構鑑定...........................................5 第四節 蛋白質序列鑑定.............................................6 第五節 質譜應用在鑑定雙硫鍵位置的方法比較.........................9 第六節 研究動機..................................................11 第七節 實驗原理..................................................12 第二章 實驗材料........................................................16 第三章 實驗方法........................................................18 第一節 蛋白質的水解 胜肽鏈的雙甲基化.............................18 第二節 Pep clean C-18 spin column 去鹽...........................19 第三節 毛細管柱的製備............................................20 第四節 HPLC 的參數設定...........................................21 第五節 質譜的參數設定............................................21 第六節 數據分析..................................................22 第四章 實驗結果與討論..................................................24 第一節 MSMS 圖譜.................................................24 第二節 一鍋與二鍋反應比較........................................25 第三節 Trypsin only 和Trypsin + Glu-C比較.......................26 第四節 問題與討論................................................27 第五章 Non-reduced的條件下對水解效率分析...............................32 第一節 研究目的..................................................32 第二節 電泳的發展................................................32 第三節 一維膠體電泳的原理........................................33 第四節 儀器與試劑................................................34 第五節 膠體配製..................................................35 第六節 結果與討論................................................36 第六章 總結與未來展望..................................................40 圖表與說明..............................................................42 參考文獻................................................................80

    (1)Yano, H.; Kuroda, S.; Buchanan, B. B. Proteomics 2002, 2, 1090.

    (2)Anfinsen, C. B.; Haber, E.; Sela, M.; White, F. H., Jr. Proc Natl Acad Sci U S A 1961, 47, 1309.

    (3)Wiley, S. R. Curr Pharm Des 1998, 4, 417.

    (4)Cook, R. B.; Rose, K. A.; Brenkert, A. L.; Ryan, P. F. Environ Pollut 1992, 77, 235.

    (5)Chan, A. C.; Carter, P. J. Nat Rev Immunol 2010, 10, 301.

    (6)Weiner, L. M.; Surana, R.; Wang, S. Nat Rev Immunol 2010, 10, 317.

    (7)Huber, R.; Deisenhofer, J.; Colman, P. M.; Matsushima, M.; Palm, W. Nature 1976, 264, 415.

    (8)Deisenhofer, J. Biochemistry 1981, 20, 2361.

    (9)Correia, I. R. MAbs 2010, 2, 221.

    (10)Dillon, T. M.; Ricci, M. S.; Vezina, C.; Flynn, G. C.; Liu, Y. D.; Rehder, D. S.; Plant, M.; Henkle, B.; Li, Y.; Deechongkit, S.; Varnum, B.; Wypych, J.; Balland, A.; Bondarenko, P. V. J Biol Chem 2008, 283, 16206.

    (11)Bragg, W. H. Science 1914, 40, 795.

    (12)Annan, R. S.; Carr, S. A. J Protein Chem 1997, 16, 391.

    (13)Gorman, J. J.; Wallis, T. P.; Pitt, J. J. Mass Spectrom Rev 2002, 21, 183.

    (14)John, H.; Forssmann, W. G. Rapid Commun Mass Spectrom 2001, 15, 1222.

    (15)Haniu, M.; Acklin, C.; Kenney, W. C.; Rohde, M. F. Int J Pept Protein Res 1994, 43, 81.

    (16)Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64.

    (17)Karas, M.; Hillenkamp, F. Anal Chem 1988, 60, 2299.

    (18)Nonami, H.; Tanaka, K.; Fukuyama, Y.; Erra-Balsells, R. Rapid Commun Mass Spectrom 1998, 12, 285.

    (19)Cleveland, D. W.; Fischer, S. G.; Kirschner, M. W.; Laemmli, U. K. J Biol Chem 1977, 252, 1102.

    (20)Mortz, E.; O'Connor, P. B.; Roepstorff, P.; Kelleher, N. L.; Wood, T. D.; McLafferty, F. W.; Mann, M. Proc Natl Acad Sci U S A 1996, 93, 8264.

    (21)Yates, J. R., 3rd; Eng, J. K.; McCormack, A. L.; Schieltz, D. Anal Chem 1995, 67, 1426.

    (22)Zhang, W.; Marzilli, L. A.; Rouse, J. C.; Czupryn, M. J. Anal Biochem 2002, 311, 1.

    (23)Chelius, D.; Huff Wimer, M. E.; Bondarenko, P. V. J Am Soc Mass Spectrom 2006, 17, 1590.

    (24)Wu, S. L.; Jiang, H.; Lu, Q.; Dai, S.; Hancock, W. S.; Karger, B. L. Anal Chem 2009, 81, 112.

    (25)Mhatre, R.; Woodard, J.; Zeng, C. Rapid Commun Mass Spectrom 1999, 13, 2503.

    (26)Fukuyama, Y.; Iwamoto, S.; Tanaka, K. J Mass Spectrom 2006, 41, 191.

    (27)Yen, T. Y.; Yan, H.; Macher, B. A. J Mass Spectrom 2002, 37, 15.

    (28)Liu, H.; Chumsae, C.; Gaza-Bulseco, G.; Hurkmans, K.; Radziejewski, C. H. Anal Chem 2010, 82, 5219.

    (29)Yen, T. Y.; Joshi, R. K.; Yan, H.; Seto, N. O.; Palcic, M. M.; Macher, B. A. J Mass Spectrom 2000, 35, 990.

    (30)Wallis, T. P.; Pitt, J. J.; Gorman, J. J. Protein Sci 2001, 10, 2251.

    (31)Zhang, B.; Cockrill, S. L. Anal Chem 2009, 81, 7314.

    (32)Choi, S.; Jeong, J.; Na, S.; Lee, H. S.; Kim, H. Y.; Lee, K. J.; Paek, E. J Proteome Res 2010, 9, 626.

    (33)Pitteri, S. J.; Chrisman, P. A.; Hogan, J. M.; McLuckey, S. A. Anal Chem 2005, 77, 1831.

    (34)Chrisman, P. A.; Pitteri, S. J.; McLuckey, S. A. Anal Chem 2005, 77, 3411.

    (35)Wang, Y.; Lu, Q.; Wu, S. L.; Karger, B. L.; Hancock, W. S. Anal Chem 2011, 83, 3133.

    (36)Huang, S. Y.; Wen, C. H.; Li, D. T.; Hsu, J. L.; Chen, C.; Shi, F. K.; Lin, Y. Y. Anal Chem 2008, 80, 9135.

    (37)Hsu, J. L.; Huang, S. Y.; Chow, N. H.; Chen, S. H. Anal Chem 2003, 75, 6843.

    (38)Hsu, J. L.; Huang, S. Y.; Shiea, J. T.; Huang, W. Y.; Chen, S. H. J Proteome Res 2005, 4, 101.

    (39)Liu, H.; Gaza-Bulseco, G.; Chumsae, C.; Newby-Kew, A. Biotechnol Lett 2007, 29, 1611.

    (40)Wu, J.; Watson, J. T. Protein Sci 1997, 6, 391.

    (41)Magagnotti, C.; Fermo, I.; Carletti, R. M.; Ferrari, M.; Bachi, A. Clin Chem Lab Med 2010, 48, 531.

    (42)Emmett, M. R.; White, F. M.; Hendrickson, C. L.; Shi, S. D.; Marshall, A. G. J Am Soc Mass Spectrom 1998, 9, 333.

    (43)Kocher, T.; Allmaier, G.; Wilm, M. J Mass Spectrom 2003, 38, 131.

    (44)Yi, E. C.; Lee, H.; Aebersold, R.; Goodlett, D. R. Rapid Commun Mass Spectrom 2003, 17, 2093.

    (45)Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551.

    (46)Zhang, Z.; Pan, H.; Chen, X. Mass Spectrom Rev 2009, 28, 147.

    (47)Kunkel, H. G.; Tiselius, A. J Gen Physiol 1951, 35, 89.

    下載圖示
    QR CODE