大部分生物抽取液(譬如：血清、胸腔液)的組成蛋白質濃度差距非常大，差距可以超過1010，也因為這非常巨大的濃度差距，那些已經被發現而且研究的非常透徹的幾個主要組成蛋白質，會遮蔽在生物抽取液中其他濃度非常低的蛋白質，使得低濃度蛋白質不容易被偵測到，因此這些低濃度蛋白質的偵測和分離是現階段蛋白質體學中一個有待克服的障礙。近年來，一些用來減少生物抽取液中各蛋白質濃度差距的方法被發展出來，在此，我們利用aminomethyl-chemmatrix微珠，因為此微珠具有兩性端(疏水端及親水端)可以去抓取蛋白質，期望可以達到平衡胸腔液樣品中各蛋白質濃度差距。我們分析處理過後的胸腔液樣品，發現那些低濃度蛋白質比較容易被二維電泳偵測到，我們接著用此方式去處理正常人的胸腔液和癌症病人的胸腔液，那些在病人和正常人中表現有差異的低濃度蛋白質較容易用二維電泳區分，而在這些有表現差異的低濃度蛋白質中，我們希望能找到屬於癌症病人的生物標記。Most of biological extracts (e.g., serum, pleural effusion) have an extremely wide dynamic concentration range of over ten orders of magnitude, so a few well-characterized, high-abundant proteins mask the signal of other low abundance proteins. The separation and detection of low abundant proteins in the proteome is still an obstacle to be solved. Many methods which can make the “hidden proteome” become easier to research are developed in recently. We report here the use of amiomethyl-chemmatrix beads to reduce the dynamic concentration range in pleural effusion (PE). The amiomethyl-chemmatrix beads have the amphiphilic nature to equalize the concentration of different protein species. After treatment with the bead, the low abundance proteins in PE are easier to detect by 2-DE PAGE. The differences in the treated PE from normal person and lung cancer person are easier determined by 2-DE PAGE and we expect that the cancer biomarkers will be discovered among the different proteomes between these two treated PE.1. Introduction…………………………………………………………………………8. Materials and methods……………………………………………………...……15 2.1. Materials……………………………………………………………….…….15.2. Methods…………………………….………………………….……………..16 2.2.1. Protein equalization by using Aminomethyl-ChemMatrix bead…....…16 2.2.2. Desalting of the eluant………………………..………………………16 2.2.3. Two-dimensional electrophoresis……………………………..……….17.2.4. In-gel digestion………………………………………………………...18.2.5. In-solution digestion…………………………………………………...19.2.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ………………………………………...………………...20. Results……………………………………………………………………………..21 3.1. The dynamic range of protein concentration in PE were decreasing after equalization technology……………………………………………………....21 3.2. The optimal loading capacity of this bead ……………………………..…......22 3.3. Application of this method to PE from normal person and lung cancer patient and compared the differences between PE…………………………………...22.4. Protein identification by LC MS/MS…………………………..……………..23. Discussion…………………………………………………………………………25. Conclusion…………………………………………………………………………27. References…………………….…….……………………………………………..28. Figures…………………………………………………………………………..…35. Tables………………………………………………………………………………4
