臺灣博碩士論文加值系統

The bioactive peptide Val-Val-Tyr-Pro (VVYP) can be genetically modified into dietary supplements which functions in reducing the triglyceride in blood and thus aid in preventing obesity, coronary heart disease and cardiovascular diseases. Dietary supplements are usually digested by various enzymes in the intestinal tract hence cause the production of functional peptides. Previous studies showed microwave-assisted system can highly improve the hydrolysis efficiency and it is timesaving. Besides, microwave-assisted method with high power usually produces lots of unpredicted miss-cleavage peptides resulting higher sequence coverage arises from longer peptides. However, this situation causes the predicted target peptides cannot be quantified. Therefore, the analysis of genetically modified VVYP storage protein of soy bean glycinin (Gy) using strategy of bi-enzyme coupled with low power microwave-assisted system is introduced to develop the best analysis platform via HPLC-ESI-SRM. By using trypsin and carboxypeptidase B (CPB), Gy1 and Gy5 will release 7 sets and 10 sets of VVYP respectively. With this strategy, a platform with high efficiency and high recovery rate for the analysis of VVYP in Gy1 and Gy5 is expected. In the future, this platform can be applied on detecting and quantifying health foods as well as protein drugs.

目錄
誌謝辭 I
中文摘要 III
Abstract IIII
目錄 V
圖表目錄 VII
第一章 緒論 1
第一節、 前言 1
第二節、 生物活性胜肽概述 2
2-1 生物活性胜肽 2
2-2 生物活性胜肽 VVYP 4
第三節、 微波輔助酵素水解分析方法及前人研究 5
3-1 微波簡介 5
3-2 微波原理 6
3-3 微波材質與吸收特性 6
3-4 前人研究 6
第四節、 質譜儀原理 7
4-1 離子源—電噴灑游離法 8
4-2 質量分析器—離子阱質量分析器 9
第五節、 串聯式質譜儀 9
第六節、 碰撞引致裂解原理 11
第七節、 研究動機 12
第八節、 研究目標 13
第二章 研究材料與方法 14
第一節、 藥品與溶劑 14
第二節、 實驗儀器與設備 15
第三節、 標準溶液製備 16
第四節、 SDS-PAGE 膠體電泳分析方法 16
第五節、 膠體 (in-gel) 和溶液 (in-solution) 酵素水解方法或搭配微波輔助 17
5-1 傳統溶液酵素水解或搭配微波輔助 17
5-2 傳統膠體酵素水解或搭配微波輔助 17
5-3 Gy 蛋白質經由膠體酵素水解和溶液酵素水解或搭配微波輔助方式 18
第六節、 標準品直接進樣 (infusion) 方式進樣質譜儀 19
第七節、 以 LC-MS/MS 方式進樣質譜儀 19
7-1 層析條件 19
7-2 傳統數據參考模式 (data-dependent) 20
7-3 選擇離子監測模式 (selected ion monitoring, SIM) 20
7-4 選擇反應監測模式 (selected reaction monitoring, SRM) 20
7-5 VVYP 檢量線製作 (calibration curve) 20
第八節、 蛋白質資料庫比對 21
第九節、 試驗動物分析 21
9-1 試驗動物材料 21
9-2 試驗動物腸道萃取方法 23
9-3 試驗動物腸道萃取 VVYP 檢測方法 23
第三章 結果與討論 24
第一節、 SDS-PAGE 膠體電泳分析 24
第二節、 以直接進樣 (infusion) 方式分析標準品 24
2-1 標準品裂解 24
2-2 標準品碰撞能量最佳化 24
第三節、 以 LC-MS/MS 製作標準品檢量線 25
第四節、 微波輔助酵素水解 VVYP 釋放量分析 25
4-1 高功率微波輔助酵素水解 VVYP 釋放量分析 25
4-2 BSA 標準品溶液 trypsin 水解測試 26
4-3 低功率微波輔助酵素水解 VVYP 釋放量分析 29
4-4 Glu-Fib 標準品溶液 CPB 水解測試 29
第五節、 試驗動物腸道萃取 VVYP 釋放量分析 30
5-1 萃取方法分析 30
5-2 試驗動物腸道萃取 VVYP 釋放量分析 30
第四章 結論 32
參考文獻 33
論文表 38
論文圖 42
論文附圖 64

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