Department of Chemistry, Materials and Chemical Engineering Giulio Natta, Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy.
J Proteomics. 2010 Feb 10;73(4):733-42. doi: 10.1016/j.jprot.2009.10.008. Epub 2009 Oct 30.
When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.
当通过组合肽配体文库捕获蛋白质时，该方法可显著降低高丰度蛋白质的浓度，并大大放大低丰度物质的信号，从而发现大量以前在蛋白质组中未检测到的蛋白质。我们一直注意到，最初存在于未处理样品中的某些物种会丢失，在某些情况下，丢失率高达 5%至 15%。这种损失令人讨厌，在一定程度上阻碍了该方法的独特性能。为了验证这些损失是否可以减少，并了解捕获过程的一些机制，我们在这里引入了捕获操作的一个重要变体，直到目前为止，该变体都是在 pH 值为 7.2 的生理盐水中进行的。在这个新方案中，结合步骤在三个不同的 pH 值下进行，即标准 pH 值 7.2，外加另外两个过程，在酸性 (pH 值 4.0) 和碱性 (pH 值 9.3) pH 值下进行。实际上，捕获过程更加广泛，在血清和其他蛋白质组中，在两个 pH 值极端条件下捕获到了许多额外的物质。有趣的是，在 pH 值 4.0 下新检测到的蛋白质大多为酸性，而在碱性 pH 值下，更多的蛋白质物质在整个等电点范围内更均匀地分布在碱性区域。讨论并评估了 pH 值在六肽文库与被分析的各种蛋白质组之间复杂结合机制中的作用。由于 pH 值会导致蛋白质图谱发生显著变化，因此建议增加通过两个示例（蛇毒和叶蛋白提取物）找到更多基因产物的可能性。通过本研究还建议控制环境 pH 值，以提高重现性研究或比较蛋白质组学分析的可能性。
