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研究生:許世勤
研究生(外文):Sh-Chien Shu
論文名稱:點帶石斑魚肌肉蛋白質體的區分,二維電泳圖譜及鑑定
論文名稱(外文):Fractionation, 2-DE Mapping and Identification of Orange-spotted Grouper's Muscle Proteome
指導教授:柯源悌
指導教授(外文):Yuan-Tih Ko
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:73
中文關鍵詞:蛋白質體學石斑魚二維電泳骨骼肌
外文關鍵詞:ProteomicsGrouper2-DESkeletal muscle
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石斑魚在海洋食品的消費及水產養殖上皆具有高度的經濟價值,特別在台灣的主要養殖與消費種類又以點帶石斑魚 (Epinephellus coioides) 為大宗。蛋白質體學方法作為用來研究石斑魚的工具,可以獲得大量關於石斑魚肌肉中所含有的生物資訊,並以此作為改善石斑魚相關產業的參考可說十分適合。基於此,本研究率先進行點帶石斑魚骨骼肌蛋白質體圖譜的建構。首先我們選擇背部白肌作為樣品,並以三種萃取方式進行區分,因此得到:總蛋白質 (T)、肌漿蛋白質 (S) 以及細胞質蛋白質 (C) 等預區分的蛋白質樣品,並進一步以一維及二維電泳進行解析。
大多數的點帶石斑魚肌肉蛋白質分布在pH 5到8之間,其中又以分子量約45 kDa的區域解析出為數最多的蛋白質點,二維電泳的膠片影像並以分析軟體ProteomweaverTM 進行比對分析。首先,比較各不同區分方法,評估何種區分較適合作為未來深入研究上的應用,並且我們也比較了兩種不同餵養方式中石斑魚肌肉的二維電泳圖譜。結果顯示總蛋白質的萃取方法可以解析出較多的差異性蛋白質點,這也表示在高油脂餵養後的石斑魚主要造成結構性蛋白產生差異性的表現。經選擇的蛋白質點經膠體內水解後以基質輔助雷射脫附離子化四極桿時間飛行式質譜 (MALDI-Q-TOF MS) 進行蛋白質的身分鑑定,得到的胜?質量指紋 (Peptide mass fingerprint, PMF) 數據利用MASCOT工具以NCBInr的非反覆性 (Nonredundant) 資料庫進行搜尋比對。將經鑑定的蛋白質特性進行分類,並對功能以及在生理及生化上所扮演的角色進行討論。藉此我們建議出未來在石斑魚蛋白質體學的深入研究上可行的方向,並盡本研究所能對改善石斑魚相
關產業的策略提出建言。
Grouper has high economic values in marine food consumption and the aquaculture industry. Especially orange-spotted grouper (Epinephellus coioides) is the major cultured and consumed species in Taiwan. Proteomics was used as a tool to collect the bio-information data of grouper muscles to be utilized in the grouper industry, by which this study begins with grouper skeletal muscle proteome mapping. We select dorsal white muscle and used three methods to fractionate muscle proteins into three protein fraction: total proteins (T), sarcoplasmic proteins (S) and cytosolic proteins (C). These pre-fractionated muscle proteins were all resolved by SDS-PAGE and two-dimensional electrophoresis (2-DE).
Most resolved protein spots distributed from pH 5 to 8, and the 45 kDa region contained the largest amount of spots. 2-DE gel images were further analyzed by ProteomweaverTM software. First, the fractionation methods were compared to estimate which one was better for further study in the future, and we compared the 2-DE map of groupers with two different rearing conditions. It showed that total protein fraction could reveal more differences, it indicates the differentiated expression maybe mainly focus on structural proteins after high lipid feeding. The interested spots were selected and for in-gel tryptic digestion, identified by MALDI-Q-TOF MS. Each peptide mass fingerprint (PMF) was searched by MASCOT in the nonredundant database of NCBInr. The character of identified proteins was classified, function and role on biochemical and physiological process were discussed. We also suggest that the possible direction for further study of grouper proteomics in the future, and mention the suggestion for grouper industry improvement as this study can do.
目錄
謝辭…………………………………………………………………......I
中文摘要……………………………………………………………….II
英文摘要………………………………………………………………IV

一、文獻回顧…………………………………………………………….1
1-1. 蛋白質體學研究…………………….…………….….2
1-2. 蛋白質體圖譜的研究……….………………….…….5
1-3. 低氧誘導研究(預口試文獻)……………………..….14
1-4. 蛋白質體學於肌肉老化方面的研究………….……20
1-5. 參考文獻………………………………….…………22
二、實驗設計………………………………….……………………….26
實驗設計流程圖…………………………………………..27
三、期刊投稿內容……………………..………………………………28
Abstract…………………………………………………...29
I. Introduction.………………………………………….…30
II. Material and Methods……………………………......32
2-1. Muscle Sample……………………………………..32
2-2. Total Protein Extraction…………………………….32
2-3. Sarcoplasmic and Cytosolic Extraction……….….32
2-4. SDS-PAGE……………………………...............…33
2-5. 2-DE…………………………………………....……34
2-6. In-Gel Digestion and MALDI-Q-TOF MS Analysis………………………………….………….34
III. Results………………………………………………...35
3-1. Gel Analysis of SDS-PAGE……………….………..35
3-2. 2-DE Gel Image Analysis…………......………......36
3-3. Further Analysis of Total Protein Fraction………..39
3-4. Protein Identification…………………………….….41
IV. Discussion………………………………………….…41
4-1. Comparison of Pre-fractionated Protein Images………......................................................41
4-2. PMF Data and MASCOT Search………………....43
4-3. Introduction of Identified Proteins………………...45
4-3-1. Creatine Kinase of Skeletal Muscle in Energy Metabolism………………………...……..………45
4-3-2. cGMP-dependent Protein Kinase to Enhance Survival Rate of Grouper……...………….….…46
V. Conclusion………………………………………….…49
VI. Literature Cited………………………………………51
四、補充探討………………………………………………………..…55
4-1. 不同餵養條件下的石斑魚………………………….56
4-2. 總蛋白質的二維電泳圖譜比對分析……………….56
4-3. 細胞質蛋白質的二維電泳圖譜比對分析……….…57
4-4. 肌漿蛋白質的二維電泳圖譜比對分析…………….58
五、附錄……………..…………………………………..…………….59
5-1. 試劑與藥品………………………………………….60
5-2. 器材與儀器………………………………………….61
5-3. 實驗流程與配方…………………………….………62
5-3-1. 總蛋白質萃取液……………………………...…..62
5-3-2. 細胞質蛋白質萃取液….…………………………..62
5-3-3. 蛋白質定量….……………………………………..63
5-3-4. SDS-PAGE………………………………………..63
5-3-5. 二維電泳 (2-DE)………………………..……….65
5-3-6. Coomassie Blue R-250 (CBR-250) 染色....……67
5-3-7. 銀染 (Silver Stain Plus)……………………..…..68
5-4. 附表….……………………………………………….70
5-5. 附圖….………………………………………………….71
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