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研究生:游雅汶
研究生(外文):Ya-Wun You
論文名稱:Halobacterium sp. NRC-1核醣體蛋白質轉譯後修飾之綜合性分析
論文名稱(外文):Comprehensive mapping of Halobacterium sp. NRC-1 ribosomal protein post-translational modification.
指導教授:吳韋訥
指導教授(外文):Wailap Victor Ng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:56
中文關鍵詞:轉譯後修飾核醣體蛋白質
外文關鍵詞:Halobacterium salinarumNRC-1Post-translational modificationPTMsribosomal protein
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在自然界中,古細菌有相較於細菌,更接近真核的蛋白質轉譯後修飾,又有相較真核較為簡單的基因體,具有商業價值的潛在商機。蛋白質轉譯後修飾( Post-translational modifications, PTMs ),在蛋白質的結構、功能、恆定上扮演重要角色。實驗上,首先將高嗜鹽古生菌Halobaterium sp. NRC-1核醣體蛋白質進行分離純化。第二,利用液相層析串聯質譜儀( LC-MS/MS )分析,不同方式進行完全或部分片斷化後的胜肽序列。並探討不同片斷化方式,所帶來的結果。第三,依據所得到的圖譜數據,辨識序列上,所進行的轉譯後修飾,其中包含甲基化( Methylation )與乙醯化( Acetylation )等修飾。至終,揭示許多蛋白質轉譯後修飾情形。
In nature, the protein modifications of archaea was closer to eukaryote than that of bacteria, and the genome of archaea is easier to manipulate than eukaryote. Therefore, archaea has potential commercial value. Post-translational modification of proteins (PTMs) play an important role in protein structure, function, and hemostasis.
Experimentally, I isolated the ribosomal proteins of a halophilic archaea, Halobaterium sp. NRC-1. Second, I applied liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyze the ribosomal proteins. Further, I compared the difference of peptide sequences identified by tradition fragmentation, and other partial fragmentation methods. Third, according to the mapping of spectra to recognize the post-translational modifications, including methylation and acetylation in the detected peptides. Finally, I revealed several ribosomal PTMs.

目錄

摘要 i
Abstract ii
目錄 iii
表目錄 v
圖目錄 vi
附錄 vi

第一章 背景介紹 1
1.1 Halobacterium sp. NRC-1簡介 1
1.2 核醣體蛋白質介紹 2
1.3 質譜技術( Tandem Mass spectrometry, MS ) 4
第二章 研究架構 7
2.1 研究動機與目的 7
2.2 實驗流程設計 8
第三章 實驗材料與方法 10
3.1 實驗材料與儀器 10
3.1.1 嗜高鹽古生菌完全培養液 (Halobacterium medium) 10
3.1.2 微量金屬溶液 (Trace metal solution) 10
3.1.3 核醣體蛋白質萃取液 (Ribosomal extraction buffer) 10
3.1.4 蔗糖濃度梯度 (Sucrose gradient) 10
3.1.5 蛋白質沉澱 (Protein precipitation) 11
3.1.6 蛋白質片斷法和去鹽化 (Protein fragmentation and desalting) 11
3.1.7 SDS-PAGE 11
3.1.8 銀染 (Silver staining) 12
3.1.9 儀器 (Apparatus) 12
3.2 分離純化嗜高鹽古生菌NRC-1之核醣體蛋白質 13
3.2.1 嗜高鹽古生菌NRC-1之菌株培養 13
3.2.2 嗜高鹽古生菌NRC-1之核醣體蛋白質純化 14
3.3 核醣體蛋白質片斷化方法 16
3.3.3 強酸部分水解法 (Acid partial hydrolysis) 17
3.3.4 微波能量輔助鹽酸部分水解法 (Microwave-assisted HCl partial hydrolysis) 18
3.4 利用蛋白質電泳、銀染法檢查水解前後之蛋白質 19
3.4.1 變性電泳 (SDS-PAGE) 19
3.4.2 銀染 20
3.5 質譜儀樣品製備與質譜儀資料分析 21
3.5.1 胜肽樣品前處理 21
3.5.2 質譜儀設定 22
3.5.3 質譜儀數據分析 22
第四章 實驗結果 24
4.1. 核醣體蛋白之蔗糖梯度分離 24
4.2. 核醣體蛋白質片斷化之結果確認 24
4.3. 質譜儀數據分析之結果 25
第五章 討論 28
參考文獻 30


圖目錄

圖1:實驗流程設計 42
圖2:核醣體蛋白質分離之情形 44
圖3:比較不同批次純化下,嗜高鹽古生菌NRC-1核醣體蛋白質樣本,其分子量圖譜分布情形 45
圖4:嗜高鹽古生菌NRC-1核醣體蛋白質之酵素片斷化 46
圖5:嗜高鹽古生菌NRC-1核醣體蛋白質之強酸水解片斷化 48
圖6:蛋白質片斷化結果比較 50
圖7:嗜高鹽古生菌NRC-1核醣體蛋白質之修飾情形 51
圖8:比較物種間核醣體蛋白質之差異 52


表目錄

表1:NRC-1核醣體蛋白質樣品 34
表2:以COMET分析方式所得之NRC-1核醣體蛋白質胜肽總數 35
表3:以PTM為搜尋分析條件所得之NRC-1核醣體蛋白質之修飾情形 (COMET) 36
表4:NRC-1核醣體蛋白質分析結果 37
表5:NRC-1核醣體S13P,在部分胰蛋白酶水解片斷法之胜肽序列 (Comet) 40
表6:更改胜肽質量容許範圍 41



附錄

附錄1:蔗糖濃度梯度製備 58
附錄2:蔗糖密度梯度離心示意圖 59



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