臺灣博碩士論文加值系統

　　環狀胜肽 (cyclic peptide) 是一種較線性多肽更為穩定且具有生理功能和醫藥價值的環狀多肽，由蛋白質或非蛋白質胺基酸藉由酯鍵、胜肽鍵、雙硫鍵所形成之環狀化合物，其廣泛分布、種類繁多且具不同功用，在鎮靜、抗菌、抗腫瘤、免疫抑制、抗酵素水解、抗化學降解、離子載體系統或蛋白質鍵結抑制物等方面展現出豐富多樣的生物活性。近年來，因化學性抗菌藥劑的長期濫用，使微生物來源的環狀胜肽變成潛在抗生素理想替代品之一，成為近期研究趨勢。目前，環狀胜肽的應用漸趨多樣化，如使用於畜牧業作為動物的飼料添加物、農業上的天然農藥、天然界面活性劑，甚至運用在高級化妝品、人類的醫學用藥等。於此以質譜分析技術為基礎，利用液相層析串聯式質譜儀 (liquid chromatography tandem mass spectrometry, LC-MS/MS) 搭配新穎裂解技術電子轉移碰撞引致裂解 (ET/CID) 達到良好定性結果，並結合選擇離子反應偵測模式 (selected reaction monitoring, SRM) 針對環狀胜肽作定量分析，在 ET/CID 定性方面，本身帶有單價數之環狀胜肽母離子，藉由鞘流溶液 (sheath liquid ) 方式添加金屬離子來提升其環狀胜肽之電荷數，提高對電子式裂解之分析效率。在 SRM 定量方面，偵測極限 (limit of detection, LOD) 在 S/N ratio > 3 為 0.41 nM ，定量極限 (limit of quantification, LOQ) 在 S/N ratio > 10 為 1.3 nM ，且變異係數 (CV%) 介於 3 ~ 11 % ；利用此分析平台可檢測出菌液中環狀胜肽含量，且線性範圍 r2 > 0.95 ；此外，針對市售牛奶與豬肝組織，皆未檢測出環狀胜肽，其線性範圍 r2 > 0.95。在本研究結果中，證實以鞘流溶液方式添加金屬離子可增加電荷數，應用於 ET/CID 此電子式裂解模式上，增加裂解資訊提高序列涵蓋率 (sequence coverage)，使定性結果擁有較高的可信度，並同時進行定量，建立一個可針對含有環狀胜肽這類化合物的 ET/CID 定性與 SRM 定量分析平台。

　　The cyclic peptide is one kind of cyclic compounds which combine of the protein and non-protein amino acids, and the carboxyl function at the C-terminus of a peptide forms a peptide bond with the N-terminal amine group a cyclic peptide is formed. A wide variety of cyclic peptides, and all has different biological functions, such as antibodies, anti-bacterial, anti-cancer, anti-immune substances, toxic substances, ion transfer regulators or protein binding inhibitor. The application of the cyclic peptide is quite diverse, for example: animal feed additive, biopesticide, surfactants, cosmetic or human drug. In the past few years, because the overuse of antibiotics, the cyclic peptides from microorganism are gradually attracted the attention of scholars. The research strategy of this experiment is based on mass analysis, using liquid chromatography tandem mass spectrometry (LC-MS/MS) combine with selected reaction monitoring (SRM) to quantitative of five cyclic peptides antibiotics (polymyxin b, colistin a, colistin b, surfactin, iturin a) in a variety of matrices (bacterial, liver and milk), and combine with electron-transfer coupled with collision-induced dissociation (ET/CID) to sequence cyclic peptides, trying to join with metal chloride solution by sheath liquid, that expect to enhance the number of electric charge for cyclic peptides in the electrospray ionization method, and observe whether increases the sequence coverage of ETD, to establishment of a cyclic peptide containing such compounds ET/CID and SRM analysis platform.

中文摘要 i
Abstract ii
目錄 iii
圖表目錄 vii
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 4
3.1 前人分析方法概述 4
3.2 金屬離子添加法輔助環狀胜肽分析 6
3.3 鞘流溶液方式添加金屬離子分析方法 7
3.4 環狀胜肽之命名系統 8
第四節 質譜儀簡介 8
4.1 離子源－電噴灑游離法 9
4.2 質量分析器－離子阱質量分析器 10
第五節 串聯式質譜儀 11
第六節 碰撞引致裂解 12
第七節 電子轉移裂解 13
第八節 電子轉移碰撞引致裂解 14
第九節 研究動機與目標 16
第二章 實驗材料 18
第一節 藥品與溶劑 18
環狀胜肽： 18
含環狀胜肽 surfactin 、 iturin a之菌液 (由弘光科技大學 - 陳珍妮老師提供)： 18
含雙硫鍵之胜肽 (內標準品) ： 18
真實樣品牛奶與豬組織： 19
金屬醋酸鹽： 19
金屬氯化鹽： 19
溶劑： 20
第二節 實驗儀器與設備 20
第三節 分析軟體 20
第三章 實驗方法 22
第一節 標準溶液的配製 22
第二節 金屬離子標準溶液的配製 22
第三節 直接進樣 (Infusion) 之上機條件 22
3.1 環狀胜肽直接進樣 (Infusion) 之上機條件 22
3.2 環狀胜肽添加金屬離子衍生化反應直接進樣 (Infusion) 之上機條件 22
3.3 以直接注入分析法 (infusion) 進樣質譜儀 23
第四節LC-MS/MS之上機條件 24
4.1 環狀胜肽標準品混合溶液 LC-MS/MS 之上機條件 24
4.2 鞘流溶液金屬離子溶液之上機條件 24
4.3 真實樣品菌液的配製與純化 24
4.4 真實樣品牛奶的配製與純化 24
4.5 真實樣品豬組織的配製與純化 25
4.6 標準添加法 25
4.7 分析方法的確效 25
4.7 以 LC-MS/MS 方式進樣質譜儀 26
第四章 結果與討論 28
第一節 以鞘流溶液添加金屬離子之條件最佳化 28
1.1 環狀胜肽介紹 28
1.2 環狀胜肽分類 29
1.3 環狀胜肽添加金屬離子 29
1.4 環狀胜肽以鞘流溶液方式添加金屬離子 30
1.5 環狀胜肽以鞘流溶液方式添加金屬離子之條件最佳化 31
1.6 鞘流溶液 dead volume 測試 31
第二節 電子轉移裂解 ETD 之條件最佳化 32
2.1 環狀胜肽添加金屬離子之 ETD 陰離子進入數量及 Act. time 條件測試 32
2.2 環狀胜肽添加之金屬離子種類比較 33
第三節 SRM 與 SETRM 定量方法比較 33
3.1 環狀胜肽 SRM 與 SETRM 定量 33
3.2 利用內標準品校正 SETRM 添加金屬離子轉換比例 35
第四節 環狀胜肽 ET/CID 定性命名 36
4.1 環狀胜肽 Polymyxin b 於不同裂解模式離子命名 36
4.2 環狀胜肽 Colistin 於不同裂解模式離子命名 37
4.3 環狀胜肽 surfactin 於不同裂解模式離子命名 37
4.4 環狀胜肽 iturin a 於不同裂解模式離子命名 38
第五節 菌液內環狀胜肽萃取方法比較 38
5.1 使用 0.22 μm 過濾膜萃取 38
5.2 使用 Oasis HLB cartridge 甲醇/乙腈 萃取 39
第六節 分析方法的確效試驗 41
6.1 準確度 41
6.2 儀器的偵測極限與定量極限 41
第七節 環狀胜肽真實樣品 ET/CID 定性與 SRM 定量 42
7.1 真實樣品菌液 42
7.2 真實樣品牛奶 43
7.3 真實樣品豬組織 (肝臟) 44
第五章 結論 46
參考文獻 47
論文表 54
論文圖 71
論文附圖 106

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