Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Nov 15;939:10-6. doi: 10.1016/j.jchromb.2013.09.008. Epub 2013 Sep 10.
Human plasma is a commonly used diagnostic fluid in clinical chemistry. In-depth plasma proteomic analysis is performed to search for disease biomarkers, however the large dynamic range of protein abundance in plasma presents a substantial analytical challenge. Removal of abundant plasma proteins using antibody capture approaches is a common and attractive means to reduce sample complexity and to aid the analysis of lower abundance proteins of interest. A novel class of heavy chain camelid-derived affinity ligands produced in Saccharomyces cerevisiae, has recently been developed as an alternative to antibody-based depletion methods. Here, we evaluate the performance characteristics of these ligands for removal of high abundance plasma proteins. Affinity ligands were tested for the removal of 14 abundant human plasma proteins. The performance characteristics were evaluated by gel-electrophoresis and LC-MS/MS of the bound and flow-through fractions. The capacity of a 5.6mL column was found to be 125μL of plasma. Replicate analysis demonstrated high column reproducibility and linearity, efficient removal of abundant proteins, and enrichment of lower abundance proteins observed after depletion. The novel class of affinity ligands provides an attractive alternative to traditional antibody-based immunodepletion methods.
人血浆是临床化学中常用的诊断液。进行深入的血浆蛋白质组学分析以寻找疾病生物标志物，然而，血浆中蛋白质丰度的大动态范围带来了实质性的分析挑战。使用抗体捕获方法去除丰富的血浆蛋白是一种常见且有吸引力的方法，可以降低样品复杂性，并有助于分析低丰度的感兴趣的蛋白质。最近，在酿酒酵母中产生了一类新型的重链骆驼科衍生的亲和配体，作为基于抗体的耗尽方法的替代方法。在这里，我们评估了这些配体用于去除高丰度血浆蛋白的性能特征。亲和配体用于去除 14 种丰富的人血浆蛋白。通过凝胶电泳和结合和流经部分的 LC-MS/MS 评估性能特征。发现 5.6mL 柱的容量为 125μL 血浆。重复分析表明柱子具有高重复性和线性、高效去除丰富的蛋白质，以及在耗尽后观察到的低丰度蛋白质的富集。新型亲和配体为传统基于抗体的免疫耗竭方法提供了一种有吸引力的替代方法。
