超音波結合鐵奈米粒子萃取微生物蛋白，比單純使用超音波萃取有更好的效果，因此我們嘗試以鐵奈米粒子為載體進行固定化酵素修飾結合超音波萃取，是否能達到同時進行萃取與消化的效果。固定化酵素具有可重複性、高穩定性及有效降低酵素自切現象等特性，加入大量固定化酵素提高酵素與蛋白質的比例，讓酵素更容易與蛋白質反應，使耗費的時間減少。本實驗以 Fe3O4@SiO2@PEI 奈米粒子利用靜電吸附的方式，得到9.061.36 pmole 胰蛋白酶/μg NPs，將固定化胰蛋白酶與未進行細胞裂解的細菌混合，利用超音波水浴結合固定化酵素鐵奈米粒子，在 37°C 水浴中超音波震盪達到萃取與消化蛋白質的效果，縮短消化時間，消化產物最後再以 LC-MS/MS 進行分析，可鑑定到 70±3 個蛋白質，與傳統細菌萃取消化方法鑑定到的 403 個蛋白質多。

目錄
壹、緒論 1
1、前言 1
2、利用質譜儀鑑定蛋白質體 2
3、胰蛋白酶 (trypsin) 的固定化 3
3.1、吸附固定法 (absorption) 4
3.2、共價鍵結法 (covalent binding) 4
3.3、包埋法 (entrapment) 5
3.4、交聯法 (cross-linking) 6
4、傳統微生物鑑定方法 7
5、細胞裂解與蛋白質萃取 8
6、質譜儀與細菌鑑定之應用 10
7、快速消化蛋白質方法 11
7.1、水浴加熱法 11
7.2、微波消化 12
7.3、超音波輔助消化 13
8、研究動機 14
貳、研究內容 15
1.、藥品與儀器 15
1.1、 藥品 15
1.2、 儀器 16
1.3、 質譜儀及液相層析儀參數與條件 17
1.4、 資料庫搜尋參數設定 19
2、實驗步驟 20
2.1、氧化鐵磁性奈米粒子合成 20
2.2、磁性奈米粒子表面修飾二氧化矽與聚乙烯亞胺 (PEI) 21
2.3、磁性奈米粒子定量 22
2.4、胰蛋白酶固定化 22
2.5、胰蛋白酶定量 23
2.6、利用牛血蛋白進行傳統水浴消化實驗 25
2.7、利用牛血清蛋白測試固定化酵素消化反應效果 25
2.8、利用 BSA 進行固定化胰蛋白酶穩定性測試 26
2.9、細菌樣品製備 26
2.10、細菌樣品萃取蛋白質 27
2.11、細菌蛋白質定量 27
2.12、細菌蛋白質水解步驟 28
2.13、固定化酵素結合超音波輔助萃取消化細菌蛋白質 29
2.14、固定化酵素萃取消化重複使用次數 30
參、結果與討論 33
1、胰蛋白酶固定化優化條件 33
1.1、Fe3O4@SiO2@PEI 磁性奈米粒子取用重量優化 33
1.2、胰蛋白酶固定時間優化 35
2、以牛血清蛋白測試固定化酵素 38
3、固定化胰蛋白酶保存時間測試 40
4、利用固定化胰蛋白酶結合超音波輔助快速鑑定微生物 43
4.1、不同重量固定化胰蛋白酶使用超音波輔助萃取消化法鑑定結果 43
4.2、固定化胰蛋白酶超音波輔助萃取消化與傳統水浴消化蛋白質結果比較 46
4.3、固定化酵素萃取消化重複使用次數 49
肆、結論 51
伍、參考文獻 53
六、附錄 59

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