臺灣博碩士論文加值系統

Chinese Abstract (中文摘要)
嗜甲烷菌中甲烷單氧化酵素可將甲烷氧化成甲醇來當它的能量及碳的來源。甲烷單氧化酵素存在兩種形式且分布在不同的細胞位置：分布於細胞質的可溶型甲烷單氧化酵素和附著於內質膜上的微粒型甲烷單氧化酵素。可溶型甲烷單氧化酵素已從許多的嗜甲烷菌中純化出來從事生化和遺傳學上的研究。然而對於微粒型甲烷單氧化酵素而言，由於當他與內質膜分離時會極不穩定，使得研究上會較困難。
微粒型甲烷單氧化酵素至少存在三個次單元，其分子量大約是45，27和23kDa。在這研究工作中，我們利用蛋白質體學技術來鑑定這些蛋白質。這個技術它結合了一維電泳與基質輔助雷射脫附游離質譜儀兩種方法且是第一次應用在微粒型甲烷單氧化酵的研究上。在電泳圖上45，27和23kDa三個位置上所顯示的次單元被鑑定為pmoB，pmoC和pmoA的基因產物。其它附著於內質膜部分的蛋白質也被分析。38 kDa位置的蛋白質被鑑定出來是pmoB基因產物經由轉譯後修飾而來的。在29kDa位置，一個名為CbbQ的蛋白質也被鑑定出來。其它較低分子量的蛋白質則是PmoB被降解的結果。
在二次串聯質譜的實驗中，我們觀察到PmoC和PmoA蛋白質N端被乙醯化。利用愛德曼降解技術，我們可以得到PmoB的N端胺基酸序列為：HGEKS。但PmoA 和PmoC由於N端被乙醯化的結果，而無法直接利用愛德曼降解技術得到N端的胺基酸序列。
利用質譜儀來分析經過胰蛋白酶在電泳膠內降解的膜蛋白質，我們發現只有露在內質膜外(比較親水)的部分能被觀察的到。基於胰凝乳蛋白酶用於電泳膠內降解蛋白質會有較多的作用位置及能得到較小的胜肽片段，因此就被選來做此實驗，並且在質譜圖中得到在內質膜內的胜肽訊號，還有較多的蛋白質範圍被獲得。
根據一些直接的資訊，可以判斷出PmoB某些胜肽部分是露在內質膜外面的。而對於PmoA和PmoC則無觀察到相同的資訊。利用這個途徑可以用來說明微粒型甲烷單氧化酵素與內質膜的相對分布。
這個研究報告了應用電泳及質譜技術來解決膜蛋白質微粒型甲烷單氧化酵素的問題。

Abstract
The enzyme methane monooxygenase (MMO), generated in methanotrophs, catalyzes the conversion of methane to methanol for use as the energy and carbon source. Two distinct forms of MMO are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO. The sMMO has been purified from several methanotrophs and has been characterized biochemically and genetically. The pMMO is less well studied than the sMMO because of the instability of the pMMO upon removal of the polypeptides from the membrane lipids.
The pMMO contains at least three subunits, of approximately 45, 27, and 23 kDa in molecular mass. In this work we identified these protein subunits with the modern proteomic tool of one-dimensional SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The three separated pMMO subunits of 45, 27, and 23 kDa were observed on the SDS-PAGE and identified as the expression gene products of the pmoB, pmoC, and pmoA, respectively. In the gel of the membrane proteins, other bands were also analyzed. The band of 38 kDa, which has been thought to be a post-translation modification product of the pmoB, appeared as a major band on the gel and identified as a PmoB fragment. A protein fragment was also observed at molecular weight 29.7 kDa, which was identified and characterized as CbbQ. The remaining the fragments appearing at low molecular weights were also identified as the PmoB fragment arising from the protease degradation.
We observed N-terminal acetylation of PmoC and PmoA by tandem mass spectrometry. The Edman degradation method was performed on the N-terminal sequences of these pMMO subunits in order to compare with the mass spectrometric results. The subunit of 45 kDa contained a sequence with HGEKS from the N terminus. Unfortunately, the other two subunits could not be directly determined by Edman degradation method due to the properties of the acetylation of these subunits.
By considering the outer membrane fraction as well as transmembrane domain, the in-gel tryptic digestion of membrane proteins could only be observed for the peptides released from the exposed domains of the membrane (more hydrophilic portions) by mass spectrometry. Due to multiple cleavage sites and smaller cleavage fragments, the chymotrypsin was chosen for in-gel digestion to cover the peptides in the membrane portion.
According to these direct information regarding cytoplasmic or transmembrane structure, we conclude that only peptides in the outer soluble portion from PmoB were observed. None of the tryptic digested peptides from the membrane bound PmoC and PmoA was observed. We have used this approach to elucidate the topological structure of the pMMO in the membrane.
This study reports the application of a method that combines modern electrophoresis with mass spectrometry to the pMMO problem may be the first case in protein level. This might well be the first application of this methodology to a membrane protein.

Table of Contents
Table of Contents 2
Abbreviations 4
Abstract 6
Chinese Abstract 8
Chapter 1: Introduction 10
Chapter 2: Experimental Methods 17
2.1 Materials 17
2.2 Growth of Methanotrophs 19
2.3 Isolation of membrane components 20
2.4 Electrophoresis and protein blotting 21
2.5 Peptide sequence determination 22
2.6 In-gel protein digestion 22
2.7 Analysis of the peptides fragments from the outer membrane protein 22
2.8 MALDI-TOF mass spectrometry 23
2.8.1 Dried droplet method. 23
2.8.2 Solution phase nitrocellulous method. 23
2.8.3 C18 ziptip. 24
2.9 MALDI-TOF mass spectrometry 24
2.10 Database searching of MALDI-TOF mass spectrometry 25
2.11 Nanoelectrospray mass spectrometry 25
2.12 Orthogonal Matrix-Assisted-Laser Desorption/Ionization (oMALDI) source of quadrupole time-of fight 26
2.13 Nanoflow ESI-MS and tandem MS (MS/MS) of Q-TOF 27
2.14 The topologies of the pMMO three subunits 27
Chapter 3: Results 31
3.1 Electrophoresis 31
3.2 Peptide mass mapping by MALDI-TOF-MS 31
3.2.1 Band P1 (45 kDa) 32
3.2.2 Band P2 (38 kDa) 33
3.2.3 Band P3, P3' (28 and 29 kDa) 33
3.2.4 Band P4 (23 kDa) 35
3.2.5 Band P5, P6 and P7 (20, 18, 14 kDa) 35
3.3 Identification of proteins using chymotrypsin for in-gel digestion 35
3.4 MS/MS sequencing by PSD or CID 36
3.4.1 MS/MS sequence of band P1, P2, P3, P3', P5 P6, P7 36
3.4.2 N-terminal modification of PmoA (P4) and Pm C (P3) 37
3.5 Analysis of N-terminal sequence of the pMMO three subunits by Edman degradation method 42
3.6 The aqueous domains of the pMMO 42
3.7 Electrophoresis without heating and analysis of MALDI-TOF-MS 43
Chapter 4: Discussion 76
4.1 Identification of three subunits 76
4.2 Comparison of Edman degradation and mass spectrometry for N-terminal sequence 77
4.3 The degradation of the pMMO 45 kDa subunit 80
4.4 Identification of the peptides at the outer membrane domain 82
4.5 Comparing the trypsin digestion and chymotrypsin digestion of the pMMO three subunits 83
Chapter 5: Conclusions 94
Chapter 6: Reference 96
Appendix 99

Chapter 6: Reference
1. Richard S. Hanson and Thomas E. Hanson (1996) Microbiological Reviews 60, 439-471
2. J. Colin Murrell, Bettina Gillbert, Ian R. McDonald (2000) Arch Microbiol 173, 325-332
3. J. D. Semrau, A. Chistoserdov, J. Lebron, A. Costello, J. Davagnino, E. Kenna, A. J. Holmes, R. Finch, J. C. Murrell, and M. E. Lidstrom (1995) Journal of Bacteriology 177, 3071-3079
4. Rosenzweig, A.C., Frederick, C. A., Lippard, S. J., and Nordlund, P. (1993) Nature 336, 537-543
5. Rosenzweig, A.C., Nordlund, P., Takahara, P. M., Frederick, C. A. and Lippard, S. J. (1995) Chem. Biol. 2, 409-418
6. Hiep-Hoa T. Nguyen, Andrew K. Shiemke, S. Joshua Jacobs, Brian J. Hales, Mary E. Lidstrom, and Sunney I. Chan (1994) J. Biol. Chem. 269, 14995-15005
7. Hiep-Hoa T. Nguyen, Sean J. Elliott, John Hon-Kay Yip, and Sunney I. Chan (1998) J. Biol. Chem. 273, 7957-7966
8. Hiep-Hoa T. Nguyen, Kent H. Nakagawa, Britt Hedman, Sean J. Elliott, Mary E. Lidstrom, Keith O. Hodgson, and Sunney I. Chan (1996) J. Am. Chem. Soc. 118, 12766-12776
9. Sergei Stolyar, Andria M. Costello, Tonya L. Peeples and Mary E. Lidstrom (1999) Microbiology 145, 1235-1244
10. Wilkins MR, Sanchez J-C, Gooley AA, Appel RD, Humphery Smith I, Hochstrasser DF, and Williams KL. (1995) Biotechnol Genet Eng Rev 13, 19-50
11. Steven P Gygi and Ruedi Aebersold (2000) Current Opinion in Chemical Biology 4, 489-494
12. Edmond DE Hoffmann, Jean Charette, and Vincent Stroobant (1996) Mass Spectrometry Principles and Application
13. Scott D. Patterson (2000) Physiological Genomics 2, 59-65
14. 50th ASMS Conference on Mass Spectrometry and Allied Topics, June 2-6, 2002, Orlando, Florida
15. Scott D. Patterson and Ruedi Aebersold (1995) Electrophoresis 16, 1791-1814
16. Bernhard Kuster and Matthias Mann (1998) Structural Biology 8, 393-400
17. France Landry, Christian R. Lombardo, and Jeffrey W. Smith (2000) Analytical Biochemistry 279, 1-8
18. Nikhil D. Phadlke, Mark P. Molloy, Stephanie A. Steinhoff, Peter J. Ulintz, Philip C. Andrews and Janine R. MAddock (2001) Proteomics 1, 705-720
19. Maya Belghazi, Katell Bathany, Codjo Hountondji, Xavier Grandier-Vazeille, Stephen Manon, Jean-Marie Schmitter (2001) Proteomics 1, 946-954
20. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cotrell, J. S. (1999) Electrophoresis 20, 3551-3567
21. Bengt Persson and Partick Argos (1994) J. Mol. Biol. 237, 182-192
22. U. K. Laemmli (1970) nature, 227, 668-685
23. Carol S. Giometti, Claudia I. Reich, Sandra L. Tollaksen, Gyorgy Babnigg, Hanjo Lim, John R. Yates Ⅲ, Gary J. Olsen (2001) Proteomics 1, 1033-1042
24. Natalia N.Nalivaeva, Anthony J. Turner (2001) Proteomics, 1, 735-747
25. Albert Stickmann, Katrin Marcus, Heike Schafer, Elke Butt-Dorje, Stefan Lehr, Armin Herkner, Silke Suer, Inke Bahr, Helmut E. Meyer (2001) Electrophoresis, 22, 1669-1676
26. Randy J.Arnold, Bogdan Polevoda, James P. Reilly, Fred Sherman (1999) J. Biol. Chem. 274, 37035-37040
27. Tomas Bergman, Madalina T. Gheorghe, Lars Hjelmqvist, Hans Jornvall (1996) FEBS, 390, 199-202
28. Michael Kinter, Nicholas E. Sherman Prorein sequencing and identification using tandem mass spectrometry
29. Alexander W. Bell, Malcolm A.Ward, Walter P. Blackstock, Hamzah N. M. Freeman, Jyoti S. Choudhary, Alan P. Lewis, Dipti Chotai, Ali FAzel, Jennifer N. Gushue, Jacques Paiement, Sandrine Palcy, Eric Cheve, Myriam Lafreniere-Roula, Roberto Solari, David Y. Thomas, Adele Rowley, and John J. M. Bergeron (2001) J. Biol. Chem. 276, 5152-5165
30. Meek, (1980) Proc. Natl. Acad. Sci. USA 77; 1632
31. James A. Zahn and Alan A. Dispirito (1996) Journal of Bacteriology, Feb 178, 1018-1029
32. I. A. Tukhvatullin, R. I. Gvozdev, and K. K. Andersson (2001), Russian Chemical Bulletin, International Edition, 50, 1867-1876
33. http://www.cbs.dtu.dk/services/SignalIP