High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins
High-abundance proteins depletion for serum proteomic analysis: concomitant removal of non-targeted proteins
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DOI：
10.1007/s00726-010-0628-x
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发表时间：
2011-01-01
期刊：
影响因子：
3.5
通讯作者：
Tomasi, Aldo
中科院分区：
文献类型：
--
作者：
Bellei, Elisa;Bergamini, Stefania;Tomasi, Aldo
在临床和药物蛋白质组学中，血清和血浆经常用于检测治疗靶标的早期诊断生物标志物。虽然获得这些体液样本是非侵入性的，而且很容易，但它们含有一些丰富的蛋白质，这些蛋白质掩盖了低浓度的其他蛋白质成分。识别血清生物标志物的挑战是去除丰富的蛋白质，同时发现和富集低丰度的蛋白质。然而，消耗策略可能导致一些可能具有潜在利益的非靶向蛋白质的同时去除。在这项研究中，我们比较了三种不同的方法，旨在从人血清中去除高丰度蛋白质，重点是识别最终可能被去除的非特异性结合蛋白质。研究了用于白蛋白去除的基于汽巴克隆蓝染料的方法、白蛋白和IgG免疫耗竭方法以及同时去除总共六种高丰度蛋白的免疫亲和柱（多亲和去除系统）。结合蛋白经洗脱、双向凝胶电泳分离、Nano LC-CHIP-MS系统鉴定。我们的结果表明，测试的方法不仅去除了高效率的靶蛋白，但也有一些非靶蛋白。我们发现，多重亲和去除柱提高了低丰度蛋白的强度，显示了新的蛋白质点，并提高了分辨率。值得注意的是，该柱显示出最低的非靶向蛋白质去除率，被证明是最有前途的去除方法，也是蛋白质组学研究前血清制备的可靠方法。
In clinical and pharmaceutical proteomics, serum and plasma are frequently used for detection of early diagnostic biomarkers for therapeutic targets. Although obtaining these body fluid samples is non-invasive and easy, they contain some abundant proteins that mask other protein components present at low concentrations. The challenge in identifying serum biomarkers is to remove the abundant proteins, uncovering and enriching at the same time the low-abundance ones. The depletion strategies, however, could lead to the concomitant removal of some non-targeted proteins that may be of potential interest. In this study, we compared three different methods aimed to deplete high-abundance proteins from human serum, focusing on the identification of non-specifically bound proteins which might be eventually removed. A Cibacron blue-dye-based method for albumin removal, an albumin and IgG immunodepletion method and an immunoaffinity column (Multiple Affinity Removal System) that simultaneously removes a total of six high-abundance proteins, were investigated. The bound proteins were eluted, separated by two-dimensional gel electrophoresis and identified by Nano LC-CHIP-MS system. Flow-through fractions and bound fractions were also analysed with the ProteinChip technology SELDI-TOF-MS. Our results showed that the methods tested removed not only the targeted proteins with high efficiency, but also some non-targeted proteins. We found that the Multiple Affinity Removal Column improved the intensity of low-abundance proteins, displayed new protein spots and increased resolution. Notably, the column showed the lowest removal of untargeted proteins, proved to be the most promising depletion approach and a reliable method for serum preparation prior to proteomic studies.