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研究生:林振平
研究生(外文):Jen-Ping Lin
論文名稱:發展多種酵素消化方法與多維高效能液相層析系統結合質譜技術對於蛋白質體之研究分析
論文名稱(外文):Development of multiple enzymatic digestion, multidimensional HPLC coupled with mass spectrometry for proteomics analysis
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shui-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:136
中文關鍵詞:質譜蛋白質體酵素消化多維液相層析
外文關鍵詞:mass spectrometrymultidimensional HPLCmultiple enzymatic digestionproteomicsprotein identification
相關次數:
  • 被引用被引用:1
  • 點閱點閱:173
  • 評分評分:
  • 下載下載:31
  • 收藏至我的研究室書目清單書目收藏:1
  本研究進行的主要目的,主要發展多種酵素消化方法以及多維的分離系統的搭配,針對真實樣品中整體蛋白質加以研究,希望藉由這些技術,提升我們蛋白質鑑定的種類與數目。蛋白質體學( proteomics )是目前最為熱門的研究課題之一。加上質譜技術的開發與進展,使我們對於生化樣品得以更進一步的深入研究,蛋白質的分析鑑定是我們此篇論文的研究重點,在有限的資料庫輔助之下,嘗試利用新的技術與分離方法,幫助我們在蛋白質上的鑑定工作。
  質輔助雷射脫附游離-飛行時間質譜法( MALDI-TOF MS ),因為其具備分析速度快、樣品需求量少、操作簡便且靈敏度高等多項優點,是本研究中我們偵測蛋白質所運用的質譜技術之一。但在質譜偵測之前,蛋白質必須先經過消化步驟,成為分子量大小不一的胜肽片斷才能適合於質譜分析。一般消化蛋白質最常用的酵素是胰蛋白酶( trypsin ),但對於較複雜的生化分子,如真實樣品,單一酵素消化有其挑戰存在:可能無法產生適合於儀器偵測範圍的片斷,或是沒有足夠的訊號以供我們分析。因此我們利用其他多種不同的酵素,分別的加入到我們欲分析的樣品中進行消化水解,藉由酵素的特異性不同,質譜可產生更多我們可以判斷的分子量訊號,最後配合蛋白質資料庫的搜尋,可使蛋白質序列的覆蓋率增加,提高鑑定出蛋白質的準確性。這裡我們以β-酪蛋白( β-casein )以及老鼠胎盤樣品為例,都可以明顯的看出多種酵素作用的優點。
  此外,在多維層析系統實驗方面( Multi-dimensional chromatogra-phy system ),針對於複雜的真實樣品,假使沒有更仔細的分離步驟,經過消化後,相對少量的微量蛋白訊號可能被抑制而無法被偵測鑑定出,因此在這裡我們也建立了一個多維的液相層析系統實驗方式,有別於一般方法在胜肽分子混合物階段才開始進行多維分離,我們在蛋白質階段就先進行初分,經過兩種酵素水解後,再搭配離子交換方法進行二維的分離分析,最後採用胜肽片斷分子量比對技術( peptide mass mapping, PMM ),雖然目前是採用離線( off-line )的階段分離來測試,但要將系統一貫地自動化動作也是指日可待。
  真實樣品最後經由基質輔助雷射脫附離子化-飛行時間質譜儀( MALDI-TOF MS )的偵測,以線上蛋白質資料庫來鑑定出所含之蛋白質種類與數目,結果顯示,應用多維系統與多種酵素方法對於真實生化樣品的分離鑑定,能夠有效提升所鑑定之蛋白質數量之外,甚至能鑑定出我們有興趣的微量功能性蛋白,這些蛋白也提供我們鑑定結果的正向佐證。期望在各項參數的最佳化、自動化後,此方法可以作為細胞、組織中整體蛋白質研究、鑑定的另一種替代技術。
  This study is mainly based on developing a multiple enzymatic digestion method and a multi-dimensional chromatography system for the analysis of biological samples. It is hoped that using this technology, further protein analysis can be identified with an increasing variety and quantity of annotated proteins. Nowadays, proteomic research is one of the hot topics in the science research field. The great invention and improvement of mass spectrometric technology allow us to extend to the next level in the research. On the main focuses of this paper is on protein identification and analysis.
  MALDI-TOF MS is one of the instrument that are used for protein analysis; its advantages include requiring small amounts of samples, short analyzing period, simple and convenient, as well as, high sensitivity. Before performing MALDI on the real sample, the proteins need to be digested into different sizes of molecules. Trypsin is the most common enzyme that is used in digesting proteins. However, due to the complexity of biological molecules, trypsin does have its limitations. If the enzyme has a hard time digesting the proteins, bad signals or even no signal would show on the screen. Consequently, different types of enzymes are added separately into each analyzing samples for digestion. Due to the fact that different enzymes have different characteristics, proteins would be digested at different digesting sites. The samples will then be detected with more signals than before. β-casein as well as rat placenta are both great examples in pointing out the advantages of using multiple enzymatic digestion method.
  Besides MALDI-TOF MS, a multi-dimensional chromatography system would meet a problem when the quantity of analyzing protein increases with a constant number of fractionations. When this happens, the signals of proteins with tiny amount would be overlapped; therefore, we have come up with a multi-dimensional high performance liquid chromatography method to resolve the problem of overlapping signals. The original multi-D LC method starts its fractionation after the proteins are digested into peptides. The new method, however, performs fractionation at the protein level. The proteins are then digested with two enzymes followed by MALDI-TOF MS. Although an off-line stage is still presently being used for detection, shifting to an automatic system is being looked forward to. The data from MS are then being searched online via protein database by peptide mass mapping (PMM) technology. This multi-dimensional fractionation method is believed to be a useful way in finding more proteins when working with actual biological samples. The method is effective to increase for identifying those proteins in micro-units, even for some low-abundantly functional protein. Hopefully with all the conditions being tuned to optimal and automatic, this method could be used to analyze and identify cells and tissues.
目錄

中文摘要..........................................Ⅰ
英文摘要..........................................Ⅲ
目錄..............................................Ⅴ
表目錄............................................Ⅹ
圖目錄............................................ⅩI

第一章 緒論
1-1前言...........................................1
1-2蛋白質體學的進展...............................5
1-2-1蛋白質體學的興起.............................5
1-2-2蛋白質的基本結構.............................7
1-2-3蛋白質的轉譯後修飾對於生物醫學方面的影響.....9
1-3蛋白質與酵素的作用.............................10
1-3-1何謂酵素.....................................10
1-3-2酵素的功能...................................11
1-3-3水解酵素的分類...............................12
1-4質譜法對於蛋白質研究的發展.....................13
1-4-1電灑游離法( ESI )的發展與應用................14
1-4-2基質輔助雷射脫附離子法( MALDI )的發展與應用..................................................15
1-5實驗架構.......................................17

第二章 利用多種酵素消化作用對於蛋白質之分析應用
2-1酵素消化於蛋白質分析上的應用...................20
2-2多種酵素消化作用探討...........................21
2-3酵素動力學介紹.................................22
2-4酵素性質及作用.................................24
2-5實驗藥品與儀器方法.............................27
2-5-1實驗樣品及試劑...............................27
2-5-2實驗酵素種類.................................28
2-5-3其他實驗儀器裝置.............................29
2-5-4質譜偵測鑑定方法及設備.......................30
2-5-5蛋白質資料庫與線上搜尋資料庫.................34
2-6實驗流程.......................................35
2-6-1標準蛋白質的測試.............................35
2-6-2真實樣品的測試...............................36
2-7結果與討論.....................................38
2-7-1實驗裝置與流程...............................38
2-7-2蛋白質標準品部分.............................38
2-7-3真實生化樣品部分.............................40

第三章 多維液相層析方法配合多種酵素消化對於真實樣品蛋白質之分析
3-1蛋白質混合物分析方法的演進.....................43
3-1-1二維膠體電泳分析方法( two-dimesional polyacryl
amide gel electrophoresis, 2D-PAGE )..............43
3-1-2多維液相層析方法於蛋白質分析的發展與應用.....46
3-2強陽離子交換方法理論...........................52
3-3多種酵素實驗的選擇.............................56
3-4實驗藥品及儀器方法.............................57
3-4-1實驗樣品與試劑...............................57
3-4-2其他儀器與裝置...............................59
3-4-3第一維階段式液相層析分離方法.................61
3-4-4第二維離子交換方法...........................63
3-4-5第三維液相層析與質譜偵測.....................65
3-5多維液相層析系統方法的進行.....................68
3-5-1標準蛋白質樣品於碳十八管柱的初步分離測試.....68
3-5-2蛋白質標準品經過trypsin消化後之胜肽分子混合物
,通過強陽離子交換管柱之分離測試..................69
3-5-3真實樣品分析部分.............................71
3-6結果與討論.....................................73
3-6-1蛋白質標準品混合物利用碳十八管柱分離結果討
論................................................73
3-6-2自行製備之強陽離子交換管柱的分離效能探討.....74
3-6-3真實樣品經過碳十八( C18 )管柱後之分離效果....75
3-6-4真實樣品的胜肽分子混合物經過強陽離子交換管
柱( SCX column )後之分段收集效果..................76
3-6-5利用胜肽片斷分子量比對技術( PMM )與多種酵素
消化作用進行各階段方法後蛋白質鑑定之結果討........78
3-6-6多維層析系統方法結合多種酵素消化作用應用於真
實生化樣品的結果討論..............................81

第四章 總結與未來展望
4-1總結...........................................84
4-1-1多種酵素水解消化方法.........................84
4-1-2多維液相層析系統方法.........................85
4-2未來展望.......................................86
參考文獻..........................................89
參考文獻
[1]:《生物資訊》生物資訊(Bioinformatics)概論,莊順淑
[2]: The Sequence of the Human Genome. SCIENCE, 291, pp.1304-1351 (2001)
[3]: International Human Genome Sequencing Consortium, Initial sequencing and analysis of the human genome. Nature, 409, pp. 860-921 (2001)
[4]: 胡湘玲,科學發展,2003年六月,366期,80-82頁。
[5]: Wolters, D. A., Washburn, M. P. & Yates, J. R. 3rd. Anal. Chem. 73,
5683–5690 (2001)
[6]: Wilkins, M.R., Sanchez, J.C., Gooley, A.A., Appel, R.D., Humphery-
Smith, I., Hochstrasser, D.F.,and Williams, K.L., Biotechnol. Genet. Eng. Rev. 13, pp. 19-50 (1996)
[7]: Hochstrasser, D.F., Clin. Chem. Lab. Med. 36, pp. 825-836 (1998)
[8]: Aebersold, R. and Goodlett, D.R., Chem. Rev. 101, pp. 269-295 (2001)
[9]: 網頁參考資料,http://www.proteomesystems.com.
[10]: 網頁參考資料,國力台灣大學生化科技系:莊榮輝教授網站http://juang.bst.ntu.edu.tw/
[11]: Edman P., Acta Chem. Scand. 4, pp.283 (1950)
[12]: Edman, P. & Begg, G., Eur. J. Biochem. 1, pp. 80–91 (1967)
[13]: Sanger F., Nobel Lecture, pp. 544-556 (1958)
[14]: Lopez, M. F. et al, J. Chromatography B, 722, pp. 191-223 (1999)
[15]: 吳昭燕,科學發展2003年10月,370期,40-47頁。
[16]: 國科會DTS計畫:國中數理教師運用網際網路輔導系統之研究
國立彰化師範大學遠距教學http://pck.bio.ncue.edu.tw/
[17]: 吳慧芬、呂麗琪《科學發展》2003年2月,362期,48∼51頁。
[18]: Yamashita M. and Fenn J. B., J. Phys. Chem., 88, pp. 4451 (1984)
[19]: Yamashita M. and Fenn J. B., J. Phys. Chem., 88, pp. 4671 (1984)
[20]: Posthumus M.A., Kistemaker P.G., and Meuzelaar H.L.C., Anal. Chem. 50, pp. 985 (1978)
[21]: Tanaka K., Waki H., Ido Y., Akita S., Yoshida Y.and T., Rapid Commun. Mass Spectrom. 2, pp. 151 (1988)
[22]: Matsuo T., Caprioli R.M., Gross M.L., Seyama Y., in: Biological Mass Spectrometry: Wiley, Chichester (1994)
[23]: Huber L. A., Nature reviews | Molecular Cell Biology, 4, pp. 74 (2003)
[24]: Blonder J., Goshe M.B., Moore R.J., Pasa-Tolic L., Masselon C.D., Lipton M.S., Smith R.D., J. Proteome res. 1, (4): pp. 351-360 (2002)
[25]: Wu C. C., Yates J. R., Nature Biotechnology, 21, (3): pp. 262-267 (2003)
[26]: van Montfort B.A., Canas B., Duurkens R., Godovac-Zimmermann J., Robillard G.T., J. Mass Spectrom. 37, (3): pp. 322-330 (2002)
[27]: van Montfort B.A., Doeven M.K., Canas B., et al., Biochimica et
Biophysica Acta. 1555, pp. 111 –115 (2002)
[28]: Gatlin, C. L.; Eng, J. K.; Cross, S. T.; Detter, J. C.; Yates, J. R., III
Anal. Chem. 72, pp. 757-763 (2000)
[29]: Quach T. T. T., Li N., Richards D. P., Zheng J., Keller B. O., Li L.,
J. Proteome res. 2 (5): pp. 543-552. (2003)
[30]: Choudhary G., Wu S. L., Shieh P., Hancock W. S., J. Proteome res. 2 (1): pp. 59-67 (2003)
[31]: Michel, H., Hunt, D. F., Shabanowitz, J. & Bennett, J., J. Biol. Chem. 263, pp. 1123–1130 (1988)
[32]: Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., Aebersold, R. Nat, Biotechnol, 17, pp. 994-999 (1999)
[33]: 網頁參考資料,http://binfo.ym.edu.tw/bch/pro/mm-kin.htm, 酵素動力學
[34]: Daniel C. L., Introduction to Proteomics-Tools for the New Bioloogy pp. 49-54. Humana Press Inc. (2002)
[35]: Khan M. A. S., Babiker E. F. E., Azakami H., Kato A., J. Agric. Food Chem. 47, (6): pp. 2262-2266 (1999)
[36]: Piszkiewicz,et al. Biochem. Biophys. Res. Commun. 40, pp. 1173
(1970)
[37]: Kraft P., Mills J., Dratz E., Analytical Biochemistry 292, pp. 76-86 (2001)
[38]: Dormady S. J., Lei J. M., Regnier F. E., J. Chromatography A, 864 (2): pp. 237-245 (1999)
[39]: Hillenkamp, F. and Karas, M. Methods Enzymol. 193, pp. 280-295 (1990)
[40]:蔡有光,Mass Spectrometry in Proteome Analysis, pp. 233-248
[41]: J.B., Mann, M., Meng, C.K., Wong, S.F., and Whitehouse, C.M.
Science 246, pp. 64-71 (1989)
[42]: Daniel C. L., Introduction to Proteomics-Tools for the New Bioloogy pp. 55~76. Humana Press Inc. (2002)
[43]: O’Farrell, P. H., J. Biol. chem. 250, pp. 4007-4021 (1975)
[44]: Wall, D. B., Kachman, M. T., Gong, S., Hinderer, R., Parus, S., Misek, D. E., Hanash, S. M., Lubman, D. M., Anal. Chem. 72 (6): pp. 1099-1111 (2000)
[45]: Liu H. J., Berger S. J., Chakraborty A. B., Plumb R. S., Cohen S. A.,
J. Chromatography B, 782 (1-2): pp. 267-289 (2002)
[46]: Giddings J.C., J. High Resolut. Chromatogr. Chromatogr. Commun.
10, pp. 319 (1987)
[47]: Giddings J.C., Anal. Chem. 56, pp. 1258A (1984)
[48]: Murphy R.E., Schure M.R., Foley J.P., Anal. Chem. 70, pp. 1585 (1998)
[49]: Davis J.M., Anal. Chem. 63, pp. 2141 (1991)
[50]: Bushey M.M., Jorgenson J.W., Anal. Chem. 62, pp. 161 (1990)
[51]: Wall D.B., Kachman M.T., Gong S.S., Parus S.J., Long M.W., Lubman D.M., Rapid Commun. Mass Spectrom. 15, pp. 1649 (2001)
[52]: Wall D.B., Parus S.J., Lubman D.M., J. Chromatogr. B Biomed. Sci. Appl. 763, pp. 139 (2001)
[53]: Chong B.E., Yan F., Lubman D.M., Miller F.R., Rapid Commun. Mass Spectrom. 15, pp. 291 (2001)
[54]: Unger K.K., Racaityte K., Wagner K., Miliotis T., Edholm L.E., Bischoff R., Marko-Varga G., J. High Resolut. Chromatogr. 23, pp. 259 (2000)
[55]: Wagner K., Miliotis T., Marko-Varga G., Bischoff R., Unger K.K., Anal. Chem. 74, pp. 809 (2002)
[56]: Wagner K., Racaityte K., Unger K.K., Miliotis T., Edholm L.E., Bischoff R., Marko-Varga G., J. Chromatography A, 893, pp. 293 (2000)
[57]: Link, A. J., Eng, J., Schieltz, D. M., Carmack, E., Mize, G. J., Morris, D. R., Garvik, B. M., Yates, J. R., 3rd. Nat. Biotechnol. 17, pp. 676-82 (1999)
[58]: Wolters, D. A., Washburn, M. P., Yates, J. R., III, Anal. Chem. 73, (23): pp. 5683-5690 (2001)
[59]: Smith R. D., Pasa-Tolic L., Lipton M. S., Jensen P. K., Anderson G. A., Shen Y. F., Conrads T.P., Udseth H. R., Harkewicz R., Belov M. E., Masselon C., Veenstra T. D., Electrophoresis, 22 (9): pp. 1652-
1668 (2001)
[60]: Qiu, Y., Sousa, E. A., Hewick, R. M., Wang, J. H., Anal. Chem. 74, (19): pp. 4969-4979 (2002)
[61]: 李建武等合編,生物化學實驗原理和方法,pp. 59~75,藝軒圖書出版社 (1999)
[62]: Robert K. S.: Protein Purification, 3rd edition, Springer-Verlag.
[63]: Yates J.R., Speicher S., Griffin P.R., Hunkapiller T., Anal. Biochem. 214, pp. 397 (1993)
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