Zellner Maria, Winkler Wolfgang, Hayden Hubert, Diestinger Michael, Eliasen Maja, Gesslbauer Bernd, Miller Ingrid, Chang Martina, Kungl Andreas, Roth Erich, Oehler Rudolf
Surgical Research Laboratories, Medical University of Vienna, Vienna, Austria.
Electrophoresis. 2005 Jun;26(12):2481-9. doi: 10.1002/elps.200410262.
For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.
为了制备用于蛋白质组分析的蛋白质，沉淀法经常被用于浓缩蛋白质并去除干扰化合物。人们应用了各种蛋白质沉淀方法，这些方法基于不同的化学原理。本研究比较了用乙醇（EtOH）或三氯乙酸（TCA）沉淀后人血小板提取物蛋白质组成的变化。两种方法从血小板制剂中获得的蛋白质量相同。然而，由于盐含量较高，乙醇沉淀的样品必须进行透析。为了鉴定单个血小板蛋白质，通过二维荧光差异凝胶电泳对样品进行分析。所有斑点中超过90%在乙醇和三氯乙酸沉淀的样品中同样存在。然而，两种沉淀方法与未沉淀样品的相关性都较小（乙醇为74.9%，三氯乙酸为79.2%）。在沉淀样品中，几种蛋白质要么减少，要么相对富集。这些蛋白质的分子量和等电点随机变化。本研究表明，蛋白质沉淀会导致蛋白质组学样品的蛋白质组成发生特定变化。这更多地取决于蛋白质的特定结构，而不是实验中使用的沉淀剂。
