An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum
An approach to remove albumin for the proteomic analysis of low abundance biomarkers in human serum
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DOI：
10.1002/pmic.200300465
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发表时间：
2003-10-01
期刊：
影响因子：
3.4
通讯作者：
Rice, G
中科院分区：
文献类型：
--
作者：
Ahmed, N;Barker, G;Rice, G
蛋白质组学技术被用来发现和鉴定与疾病相关的生物标志物。这些技术在患者血清中寻找潜在诊断/预后生物标志物的应用受到了高丰度白蛋白和免疫球蛋白的限制，这些白蛋白和免疫球蛋白约占总血清蛋白的 60-97%。本研究的目的是评估在二维凝胶电泳 (2-DE) 分析之前单独使用 Affi-Gel Blue 或与 Protein A（Aurum 血清蛋白迷你试剂盒，Bio-Rad）联合处理人血清是否可以去除高丰度蛋白质，从而使低丰度蛋白质可视化。血清样品用 Affi-Gel Blue 或 Aurum 试剂盒处理，然后使用 11 cm、pH 4-7 等电聚焦条进行 2-DE（第一维）和 10% 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（第二维）。使用荧光蛋白染料（SYPRO Ruby，Bio-Rad）使蛋白质斑点可视化。处理方法之间的比较表明，Affi-Gel Blue 和 Aurum 试剂盒均显着去除了白蛋白，并且每次处理后凝胶的蛋白质谱存在显着差异。处理之间的直接比较揭示了 Affi-Gel Blue 特有的 28 个蛋白质点，而 Aurum 试剂盒处理后只有 2 个点是独特的。 Affi-Gel Blue 和 Aurum 试剂盒处理的血清中的独特斑点在未处理的血清中未显现出来。经过 16 小时的 Affi-Gel Blue 处理，53 个蛋白点的可视化效果增强了两倍、31 个蛋白点增强了 5 倍、12 个蛋白点增强了 10 倍、6 个蛋白点增强了 20 倍。同时，在 Aurum 试剂盒处理后，观察到 30、13、8 和 5 个蛋白质点分别增强了 2 倍、5 倍、10 倍和 20 倍。两种处理方法中蛋白质斑点可视化增加的模式相似。总之，在 2-DE 分析之前使用 Affi-Gel Blue 或 Aurum 试剂盒处理血清样品可用于去除高丰度蛋白质，以提高低丰度蛋白质的检测灵敏度。
Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by twofold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before 2-DE analysis can be used to remove high abundance proteins in order to increase the detection sensitivity of proteins present in low abundance.