Granger Jill, Siddiqui Javed, Copeland Shannon, Remick Daniel
Department of Pathology, University of Michigan, 2210 Medical Science I Building, 1301 Catherine Street, Ann Arbor, MI 48109-0602, USA.
Proteomics. 2005 Dec;5(18):4713-8. doi: 10.1002/pmic.200401331.
The use of proteomics for efficient, accurate, and complete analysis of clinical samples poses a variety of technical challenges. The presence of higher abundance proteins in the plasma, such as albumin, may mask the detection of lower abundance proteins such as the cytokines. Methods have been proposed to deplete the sample of these higher abundance proteins to facilitate detection of those with lower abundance. In this study, a commercially available albumin depletion kit was used to determine if removal of albumin would measurably reduce detection of lower abundance cytokine proteins in human plasma. The Montage Albumin Deplete Kit (Millipore) was used to deplete albumin from LPS-stimulated whole blood from 15 normal human donors. Albumin depletion was measured using the BCG reagent and SDS-PAGE, and cytokine recovery was determined by a microassay immunoassay that measures both pro- and anti-inflammatory cytokines. Average albumin depletion from the samples was 72%. However, several cytokines were also significantly reduced when the albumin was removed from the plasma. Additionally, there was a variable reduction in cytokine recovery from a known mixture of cytokines in a minimal amount of plasma that were loaded onto the columns. These data demonstrate that there may be a non-specific loss of cytokines following albumin depletion, which may confound subsequent proteomic analysis.
使用蛋白质组学对临床样本进行高效、准确和全面的分析面临着各种技术挑战。血浆中存在较高丰度的蛋白质，如白蛋白，可能会掩盖较低丰度蛋白质（如细胞因子）的检测。已经提出了一些方法来去除样本中这些较高丰度的蛋白质，以促进对较低丰度蛋白质的检测。在本研究中，使用一种市售的白蛋白去除试剂盒来确定去除白蛋白是否会显著降低人血浆中较低丰度细胞因子蛋白的检测。使用蒙太奇白蛋白去除试剂盒（密理博公司）从15名正常人类供体的脂多糖刺激的全血中去除白蛋白。使用BCG试剂和SDS-PAGE测量白蛋白去除情况，并通过一种可同时测量促炎和抗炎细胞因子的微量免疫测定法确定细胞因子回收率。样本中白蛋白的平均去除率为72%。然而，当从血浆中去除白蛋白时，几种细胞因子也显著减少。此外，将已知的细胞因子混合物以少量血浆加载到柱上时，细胞因子回收率存在可变程度的降低。这些数据表明，白蛋白去除后可能存在细胞因子的非特异性损失，这可能会混淆后续的蛋白质组学分析。
