蛋白質體學(proteomics)是後基因時代(post genomic era)研究的重點之一。其中之一的研究就是細胞中蛋白質的篩選(screen)與鑑定(identification)。目前用來進行此項工作最重要的生化技術之一就是結合2-D膠體電泳及基質輔助雷射脫附游離質譜法(MALDI/MS)。
本研究利用2-D膠體電泳分離血清中的蛋白質再以酵素進行消化分解，所得之胜肽隨後以MALDI進行分析，再配合資料庫搜尋，即可在短時間鑑定出微量蛋白質。研究結果發現當分析物為蛋白質混合物時，在經酵素分解後，並以MALDI進行分析時，往往來自蛋白質含量較高的胜肽訊號會壓抑來自蛋白質含量較低的胜肽訊號，進而使得在資料庫搜尋時無法正確找到低含量之蛋白質。為解決此項缺失，本研究探討了幾項可能的解決方法：一、利用更換MALDI基質方式以選擇性的提高來自低含量蛋白質的胜肽訊號，二、改變進行資料庫比對時的參數設定，以利微量蛋白質的搜尋，三、使用其他酵素，不同專一性的酵素會切在不同的胺基酸上，對同一蛋白質會切出不同的胜肽圖譜，四、以游離源後碎裂（post source decay; PSD）方式進行來自不同蛋白質胜肽的序列分析以提高鑑識率。

Detection trace compound from human serum by matrix-assited laser desorption ionization mass spectrometry and second dimensional gel ectrophoresis

目錄
摘要……………………………………………………………………Ⅰ

目錄…………………………………………………………………….Ⅱ

圖目錄………………………………………………………………… Ⅳ

表目錄.....................................................................................................Ⅵ


壹、 緒論…………………………………………………………….1
一、 前言…………………………………………………………..1
二、 基質輔助雷射脫附游離質譜法(MALDI)…………………..4
1. 發展歷史……………………………………………………4
2. 樣品製備……………………………………………………4
3. 飛行時間偵測器……………………………………………5
(1) 直線型TOF……………………………………………6
(2) 反射型TOF……………………………………………7
4. MALDI中離子的形成機制……………………………….9
5. MALDI中基質的特性與功能…………………………….9
(1) 吸收並轉移雷射能量…………………………………10
(2) 隔絕分析物……………………………………………10
(3) 提供氫離子……………………………………………10

三、 電泳分離法…………………………………………………12
1. 發展歷史………………………………………..…………12
2. 電泳的操作原理………………………………..………….15
3. 二維膠體電泳…………………………………...…………17
(1) 一維等電點電法………………………………………17
(2) 二維SDS-PAGE膠體電泳……………………………18

(3) In gel digestion…………………………………………18

四、 人類血液的組成…………………………………………….21
五、 論文目標…………………………………………………….23

貳、 實驗…………………………………………………………....25
一、 儀器裝置……………………………………………………25
二、 試劑配製及實驗操作………………………………………28

參、 結果與討論……………………………………………………44

肆、 結論……………………………………………………………75

伍、 參考文獻………………………………………………………76

圖目錄

圖1 直線型及反射型飛行時間質譜儀……………………………….8
圖2 丙烯醯胺聚合作用之反應式……………………………………14
圖3 Bruker omniFLEX-RE-MALDI-TOF儀器構造圖……………...26
圖4 將IEF-strip放到SDS-PAGE上進行二維分離…………………36
圖5 In gel digestion之示意圖………………………………………..41
圖6 為 (a) Cytochrome C (b) Albumin (c) Transferrin 經過trypsin digestion後之MALDI質譜圖…………………………………46
圖7 Cytochrome C、Albumin、Transferrins三種蛋白質混合後
，再經由trypsin digestion後所得之MALDI質譜圖…………48
圖8 (a)血清與(b)dealbumin血清之MALDI質譜圖………………49
圖9 血清之2-D 膠體電泳圖……………………………………….51
圖10 為圖9中之 (a) A點 (b) B點 (c) C點，經過 in gel
digestion之MALDI質譜圖……………………………………52
圖11 2-D膠體電泳以不同染色法所得之電泳圖…………………...55
圖12 以銀染色法所得之2-D膠體電泳圖…………………………..56
圖13 Human Albumin經過 in gel digestion 所得之MALDI質圖…57
圖14 將圖12之A點經過in gel digestion所得之MALDI質譜圖..58
圖15 將圖12之B點經過in gel digestion所得之MALDI質譜圖…59
圖16 將圖12之C點經過in gel digestion所得之MALDI質譜圖…60.
圖17 將圖12之D點經過in gel digestion所得之MALDI質譜圖….61
圖18 將圖12之E點經過in gel digestion所得之MALDI質譜圖….62
圖19 將圖12之F點經過in gel digestion所得之MALDI質譜圖…..63
圖20 將圖12之G點經過in gel digestion所得之MALDI質譜圖….64
圖21 為Cytochrome C以trypsin digestion的時間分別在
(a) 0 sec(b) 30 sec (c) 1 min (d) 5 min (e) 10 min 所得
(b) 之MALDI質譜圖………………………………………….67
圖22 為Myoglobin以trypsin digestion的時間分別在
(a) 0 sec(b) 30 sec (c) 1 min (d) 5 min (e) 10 min 所得
(b) 之MALDI之質譜圖………………………………………..68
圖23 將Cytichrome C及Myoglobin以不同比例混合
(a) 1:1 (b) 1:5 (c) 1:10 (d) 5:1 (e) 10:1, 再以trypsin
digestion 所得之MALDI質譜圖…………………………….70
圖24 為Cytochrome C經過trypsin digestion後，以不同
基質分析之MALDI質譜圖………..………………………….72
圖25 為Myoglobin經過trypsin digestion後，以不同基
質分析之MALDI質譜圖………………………………………74

表目錄
表1 資料庫搜尋結果 ………………………………..................... 65
表2 改變搜尋參數 ……………………………………................. 71

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