A Systematic Analysis of Eluted Fraction of Plasma Post Immunoaffinity Depletion: Implications in Biomarker Discovery

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at血浆是临床蛋白质组学（clinical proteomics）中用于生物标志物（biomarker）发现的最易获取的样本来源。然而，由于蛋白质组具备极宽的动态范围，从血浆中筛选潜在生物标志物仍是一项颇具挑战性的工作。血浆中的潜在生物标志物通常丰度极低，因此要鉴定这类低丰度蛋白质，必须先去除高丰度蛋白质。采用多亲和去除系统（Multi-Affinity Removal System, MARS）对高丰度蛋白质进行免疫去除以实现样本预分级，是当前去除多种高丰度蛋白质的主流方法。但这类高丰度蛋白质的去除过程，可能会伴随低丰度蛋白质的非特异性丢失。尽管已有部分研究报道存在非靶标蛋白质被去除的情况，但学界主流观点认为这类非靶标蛋白质的数量较少。本研究中，我们对与靶标高丰度蛋白质一同被去除的蛋白质进行了鉴定。本研究使用三款MARS亲和柱（Hu-6、Hu-14及Proteoprep 20）分别对三份血浆样本开展高丰度蛋白质去除处理。将亲和结合组分采用LTQ-Orbitrap质谱仪进行gelC-MS分析。本研究采用包括自主开发的MassWiz在内的四款数据库搜索算法，对鉴定得到的肽段进行筛选。