Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany.
Mol Cell Proteomics. 2010 Jun;9(6):1271-80. doi: 10.1074/mcp.M900419-MCP200. Epub 2010 Feb 16.
Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses.
抗体微阵列有可能实现对少量样本材料的全面蛋白质组学分析。在这里，我们提供了使用双色模式生产、质量评估和可重复应用抗体微阵列的方案，该微阵列包含 1800 个特征，代表了 810 种针对 741 种癌症相关蛋白的抗体。除了评估阵列质量的措施外，我们还实施了用于双重颜色检测准确性和显著性的指标。与单颜色方法相比，双色测量在检测重复性和区分能力方面表现出色。在血清样本分析中，高丰度蛋白的耗竭并没有提高技术检测质量。相反，耗竭会导致蛋白质表达产生强烈的偏差。在初步研究中，我们证明了该方案在尿液样本中衍生蛋白质的适用性。我们鉴定了胰腺癌患者和健康受试者以及不同性别的尿液样本之间的差异。这项研究表明可以生成与生物医学相关的数据。通过全面的质量分析表明，双色抗体阵列方法与其他蛋白质组学技术具有竞争力，并且在性能上与转录组微阵列分析相当。
