Proteomic analysis of human plasma proteins by two-dimensional gel electrophoresis and by antibody arrays following depletion of high-abundance proteins
Proteomic analysis of human plasma proteins by two-dimensional gel electrophoresis and by antibody arrays following depletion of high-abundance proteins
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DOI：
10.1007/s12013-007-0048-z
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发表时间：
2007-01-01
影响因子：
2.6
通讯作者：
Beliveau, Richard
中科院分区：
文献类型：
--
作者：
Desrosiers, Richard R.;Beaulieu, Edith;Beliveau, Richard
检测人类血浆中存在于较低水平的蛋白质，以识别潜在的疾病生物标记物，由于少数高度丰富的蛋白质而变得复杂。一个有希望的策略是去除这些丰富的蛋白质，干扰蛋白质组技术对血浆含量的分析。这项研究比较了三种基于亲和力的方法来去除人体血浆中最丰富的蛋白质。其中两种是基于抗体，这种抗体耗尽了6种或12种最丰富的蛋白质，在富集度较低的血浆蛋白方面表现出了最高的效率。EDTA和CTAD两种抗凝剂对血浆制备的比较表明，这种处理影响了二维(2D)凝胶上可见的低丰度蛋白质的模式。几种染色方法，包括两种荧光染料，Sypro红宝石和深紫色，也与一种非常灵敏的银染色法进行了比较，以显示二维凝胶上的低丰度蛋白质。此外，用羟乙基二硫化物处理低丰度的血浆蛋白提高了蛋白质的清晰度和分辨率。所有这些系统比较的目的是在血浆制备和处理的不同步骤以及在二维凝胶分析蛋白质时选择最可靠的方法，以获得高重复性的低丰度血浆蛋白图谱。重要的是，通过这些优化条件浓缩的低丰度血浆蛋白被抗体微阵列进一步分析，允许使用350个针对信号蛋白的抗体来鉴定61个蛋白，这表明这种蛋白质组学策略是检测潜在的血浆生物标志物的一种有价值的方法。
Detecting proteins that are present at lower levels in human plasma, for the identification of potential disease biomarkers, is complicated by a few highly abundant proteins. One promising strategy is the removal of these abundant proteins interfering with the analysis of plasma content by proteomic techniques. This study compared three affinity-based methods to remove the most abundant proteins in human plasma. Two of them, based on antibodies, which depletes the six or the 12 most abundant proteins, demonstrated the highest efficiency in enriching less abundant plasma proteins. Comparison of two anticoagulant treatments for plasma preparation, EDTA and CTAD, showed that this treatment influenced the patterns of lower-abundance proteins visible on 2-dimensional (2D) gels. Several staining procedures including two fluorescent dyes, Sypro Ruby and Deep Purple, were also compared with a very sensitive silver staining method for the visualization of lower-abundance proteins on 2-D gels. Further-more, treatments of lower-abundance plasma proteins with hydroxyethyl disulfide enhanced protein sharpness and resolution. The purpose of all these systematic comparisons was to select the most reliable methods in different steps of plasma preparation and handling as well as in analysis of proteins by 2-D gels to obtain highly reproducible patterns of lower-abundance plasma proteins. Importantly, the lower-abundance plasma proteins enriched by these optimized conditions were further analyzed by antibody microarrays allowing the identification of 61 proteins using 350 antibodies directed against signalling proteins suggesting that this proteomic strategy is a valuable approach for detecting potential plasma biomarkers.