三氯乙酸/丙酮蛋白质提取法在白内障晶状体蛋白提取中的应用

Application of TCA/acetone protein extraction in the cataract lens protein extract

摘要:

目的：比较不同蛋白质提取方法在白内障晶状体蛋白提取中的效果，寻找适合白内障晶状体蛋白质组学研究的蛋白质提取方法。

方法：采用裂解液溶解白内障晶状体组织样本，超声裂解离心后分别采用三氯乙酸/丙酮法和ReadyPrep 2D Cleanup Kit对晶状体蛋白样本进行提取、二维电泳，并对电泳后凝胶进行染色、图像采集和图像分析。

结果：在2-DE图谱上蛋白斑点的相对分子量在14～97.4kDa之间，等电点在5～9之间，其中高丰度蛋白相对分子量处于20～31kDa范围内，低丰度蛋白斑点相对分子量处于31～43kDa范围内。经TCA/丙酮沉淀法处理的含有透明质酸钠白内障晶状体蛋白样本，二维电泳凝胶图谱背景较清晰，蛋白斑点较清楚，共检测到35～40个左右的蛋白斑点。

结论：在人白内障晶状体蛋白质组学研究中，TCA/丙酮法具有较好的纯化效果，为人晶状体组织蛋白质组学的质谱分析研究提供了可靠的二维电泳凝胶图谱。

Abstract:

AIM: To compare the effectiveness of different protein extraction method in the cataract lens protein extract, and look for the best protein extraction method suitable for cataract proteomics research.

METHODS: Cataract lens samples were dissolved in lysis buffer and sonication; the samples were extracted by TCA/acetone and protein extraction kit after centrifugation. Lens proteins of the samples were analyzed by two-dimensional electrophoresis(2-DE), gel electrophoresis and staining, image acquisition and image analysis.

RESULTS: According to the results obtained from two-dimensional electrophoresis gel image, the molecular weight of protein points were localized at 14-97.4kDa, PI5-9. The molecular weight of high abundance crystallins were localized at 20-31kDa, the molecular weight of lower abundance crystallins were localized at 35-45kDa. The 2-DE image background of cataract lens sample with hyaluronic acid extracted by TCA /acetone was clear, no obvious vertical bands, point of proteins were more clear, and 35 to 40 protein points were detected.

CONCLUSION: TCA/acetone extraction method has better purification compared to other methods in human cataract lens proteomics, and provides the reliable two-dimensional polyacrylamide gel Image for human cataract lens proteomics mass spectrometry analysis.