Department of Biology, Functional Genomics and Proteomics Group, KU Leuven, Naamsestraat 59, 3000 Leuven, Belgium.
VITO Vlaamse Instelling voor Technologisch Onderzoek, Boeretang 200, 2400 Mol, Belgium.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Jul 1;1089:43-59. doi: 10.1016/j.jchromb.2018.04.045. Epub 2018 May 1.
Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.
血浆是生物标志物研究的一种极具价值的资源，因为它易于获取，并且包含了大量有关患者健康状况的信息。尽管蛋白质组学领域的进步使得能够分析血浆蛋白质组，但由于其高度复杂性和较大的动态范围，鉴定低丰度蛋白质仍然具有挑战性。为了降低蛋白质浓度的动态范围，开发了一种串联耗竭技术，该技术由硫酸铵沉淀和 Protein A 亲和层析组成。使用这种方法，在 20%至 40%的硫酸铵饱和度水平下，从血浆样品中去除了 50%的白蛋白，以及其他高丰度蛋白质，如α-1-抗胰蛋白酶。与使用 Protein A 柱去除免疫球蛋白结合使用时，当进行 shotgun 质谱分析时，可鉴定多达 40 种新的低丰度至中丰度蛋白质。与未耗竭的血浆相比，在 2D-PAGE 分析中观察到 270 个额外的蛋白质斑点。这些结果表明，这种串联耗竭方法与基于免疫亲和层析的商业试剂盒相当。此外，使用 Protein A 免疫球蛋白耗竭的这种方法显示出高度可重现性，并且去除了很少量的非靶蛋白。硫酸铵沉淀和 Protein A 亲和层析的组合提供了一种低成本、高效、简单且可重现的替代商业试剂盒的方法，蛋白质保持天然构象，允许进行蛋白质活性和蛋白质相互作用研究。
