不同等电点沉淀法和超速离心法提取牛奶乳清蛋白的双向电泳分析

2-DE Analysis of Whey Protein Extracted by Different Isoelectric Precipitations and Ultracentrifugation Methods from Cow Milk

为探索牛奶乳清蛋白提取的最适方法,以生鲜荷斯坦牛奶为对象,采用不同等电点(pH 4.6和pH 4.8)沉淀法和超速离心法提取牛奶乳清蛋白样品,并对提取效果进行双向凝胶电泳(two-dimensional electrophoresis,2-DE)图谱分析。依据凝胶图谱上蛋白斑点比较图谱质量,采用PDQuest 8.0凝胶图像分析软件进行凝胶图谱分析。结果显示:2种方法均能有效提取牛奶乳清蛋白,且获得的2-DE凝胶图谱背景清晰、蛋白点个体独立、无明显的拖尾现象,且重复性强,但都存在一定的酪蛋白残留。与超速离心法相比,pH 4.6沉淀法和pH 4.8沉淀法提取乳清蛋白的2-DE凝胶图谱可检测到较多的蛋白质斑点。pH 4.6沉淀法提取乳清蛋白制备的2-DE凝胶图谱中蛋白点的表达丰度略高于pH 4.8沉淀法。研究表明:pH 4.6沉淀法提取乳清蛋白制备2-DE凝胶图谱略优于pH 4.8沉淀法和超速离心法。但2种等电点沉淀法和超速离心法都可有效去除牛奶中的高丰度酪蛋白,提高低丰度蛋白的检出敏感性。

The objective of this study was to investigate the effect of extraction methods on two-dimensional electrophoresis (2-DE) maps of whey proteome in cow milk and to explore an optimal extraction method. Samples were extracted from cow milk by different isoelectric precipitations and ultracentrifugation methods. 2-DE maps were analyzed by PDQuest 8.0 software. The results of 2-DE maps showed that milk whey protein could be effectively extracted by the above methods with less background, no significant strips and good repeatability. However, there were still some residual caseins appearing in each map. 2-DE maps of whey protein refined by isoelectric precipitations were relatively informative when compared with the gel maps obtained by ultracentrifugation. Moreover, the richness of different whey proteins in various maps extracted by adjusting pH to 4.6 as isoelectric point was slightly higher than adjusting pH to 4.8. The results indicated that adjusting pH to 4.6 as isoelectric point to extract whey protein had some advantages than adjusting pH to 4.8 and ultracentrifugation. However, all the methods used in this study could effectively remove high abundant casein to improve the detection sensitivity of low abundance proteins.