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研究生:曾玉亨
研究生(外文):Yu-Heng Tseng
論文名稱:膜蛋白質體學:利用質譜技術進行嗜甲烷菌中微粒體甲烷單氧化酵素之分離與序列分析
論文名稱(外文):Membrane Protein Proteomics: Isolation and Sequence Analysis of the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) by Modern Mass Spectrometry
指導教授:陳長謙陳長謙引用關係陳玉如陳玉如引用關係
指導教授(外文):Sunney I. Chan, Ph. DYu-Ju Chen, Ph. D
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學系
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:2002
畢業學年度:90
語文別:英文
論文頁數:111
中文關鍵詞:微粒型甲烷單氧化酵素嗜甲烷菌基質輔助雷射脫附游離質譜儀甲烷單氧化酵素二次串聯質譜
外文關鍵詞:pMMOmethanotrophsMALDI-TOF-MSMMOMS/MS
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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
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