臺灣博碩士論文加值系統

環狀胜肽 (cyclic peptide) 是由蛋白質或非蛋白質胺基酸藉由胜肽鍵所形成之環狀化合物，其種類繁多，並各自有不同功用，如可作為抗體、毒性物質、抗免疫物質、離子傳導調控物質、蛋白質鍵結抑制物或酵素抑制物。目前，環狀胜肽的應用相當多樣化，如使用於畜牧業作為其動物的促進生長藥物、農業上的天然農藥、天然界面活性劑等，甚至運用在高級化妝品、土壤復育、原油回收、幫助去除重金屬汙染等。對於環狀胜肽的相關研究，逐漸吸引學者的關注，本實驗的研究策略是以質譜分析技術為基礎並有別於前人研究以碰撞誘導裂解 (collision induced dissociation, CID) 分析環狀胜肽為主，嘗試運用其他較為新穎的裂解技術，包含電子轉移裂解 (electron transfer dissociation, ETD)、Electron-transfer collisionally activated dissociation (ETcaD) 及電子轉移結合碰撞引致裂解 (electron-transfer coupled with collision-induced dissociation, ET/CID) 進行環狀胜肽定性技術開發與探討。對於本身較不易帶有多價數之環狀胜肽，嘗試藉由添入金屬醋酸鹽溶液提升環狀胜肽於電灑法中電荷數，並觀察其是否可提升 ETD 的裂解效率，增加裂解資訊提高序列涵蓋率 (sequence coverage)，使其擁有較高的可信度，建立一套環狀胜肽之質譜分析平台，協助未來相關菌種純化環狀胜肽之品質監測。

The cyclic peptide is one kind of cyclic compounds which combine of the protein and non-protein amino acids, many of kinds has more different functions, such as antibodies, toxic substances, anti-immune substances, ion transfer regulators, protein binding inhibitor or enzyme inhibitors. The application of the cyclic peptide is quite diverse, and gradually attracted the attention of scholars. The research strategy of this experiment is based on electron-spray ionization tandem mass spectrometry (ESI-MS/MS) combine with collision induced dissociation (CID) and electron transfer dissociation (ETD) to sequence cyclic peptides, to apply to the determination of cyclic peptides, Surfactin、Colistin、Polymyxin B、Iturin a and Bacitracin. Observations of different amino acid residues from CID and ETD experiments for the peptide were interpreted by their fragments, and try to join with metal acetate solution, that expect to enhance the number of electric charge for cyclic peptide in the electrospray ionization method, and observe whether increases the sequence coverage of ETD or not, to improve the qualitative degree the establishment of a cyclic peptide analysis platform, it might be the related bacteria purified cyclic peptide technology development and quantitative research.

中文摘要..........................i
Abstract........................ii
目錄............................iii
圖表目次..........................v
第一章 緒論........................1
第一節 前言........................1
第二節 環狀胜肽簡介 .................2
第三節環狀胜肽相關分析方法............3
3.1前人分析方法概述.................3
3.2金屬離子添加法輔助環狀胜肽分析......4
第四節 質譜儀簡介...................6
4.1 離子源-電噴灑游離法..............6
4.2 質量分析器-離子阱質量分析器........7
第五節 串聯式質譜儀..................8
第六節 碰撞引致裂解..................9
第七節 電子轉移裂解..................10
第八節 ETcaD 模式...................11
第九節 電子轉移碰撞引致裂解............11
第十節 研究動機與目標.................12
第二章 實驗部分......................14
第一節 藥品與溶劑....................14
環狀胜肽:...........................14
金屬醋酸鹽:.........................15
金屬氯化鹽:.........................16
溶劑:..............................16
第二節 實驗儀器與設備.................17
第三節 分析軟體......................17
第三章 實驗方法......................18
第一節 標準溶液的配置.................18
第二節 金屬離子溶液的配置..............18
第三節 環狀胜肽以 infusion 之上機條件..18
第四節 金屬離子衍生化反應..............18
第四節 以直接注入分析法(infusion)進樣質譜儀...19
第四章 結果與討論.....................20
第一節 前處理最佳化...................20
1.1 環狀胜肽分類.....................20
1.2 金屬離子的添加....................20
1.3 環狀胜肽添加金屬離子之條件測試-反應溫度....21
1.4環狀胜肽添加金屬離子之條件測試-ETD反應時間..22
第二節 直接進樣 (infusion)分析 – 環狀胜肽特性介紹...23
第三節 環狀胜肽質譜鑑定與探討............24
3.1 裂解模式於正二價環狀胜肽母離子分析....25
3.2 裂解模式於正二價環狀胜肽母離子添加金屬離子之分析...29
3.3裂解模式於帶正一價環狀胜肽母離子分析...30
3.4 裂解模式於正一價環狀胜肽母離子添加金屬離子之分析...31
3.4.1 環狀胜肽添加醋酸鹽之結果探討.......32
3.4.2 環狀胜肽添加氯化鹽之結果探討.......36
3.4.3針對surfatin添加金屬離子的切位比對確認與裂解趨勢...39
3.4.4 ET/CID 於 iturin a添加金屬離子後分析之優點.....40
3.5裂解模式於特殊結構環狀胜肽母離子添加金屬離子之分析.....40
結論................................44
參考文獻.............................46
論文表格.............................52
論文圖...............................73
論文附圖.............................233

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