Laboratory of Biopharmaceutical Research, National Institute of Biomedical Innovation, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
Biomaterials. 2011 Jan;32(1):162-9. doi: 10.1016/j.biomaterials.2010.09.030. Epub 2010 Oct 8.
Proteomics-based analysis is currently the most promising approach for identifying biomarker proteins for use in drug development. However, many candidate biomarker proteins that are over- or under-expressed in diseased tissues are found by such a procedure. Thus, establishment of an efficient method for screening and validating the more valuable targets is urgently required. Here, we describe the development of an "antibody proteomics system" that facilitates the screening of biomarker proteins from many candidates by rapid preparation of cross-reacting antibodies using phage antibody library technology. Using two-dimensional differential in-gel electrophoresis analysis, 16 over-expressed proteins from breast cancer cells were identified. Specifically, proteins were recovered from the gel pieces and a portion of each sample was used for mass spectrometry analysis. The remainder was immobilized onto a nitrocellulose membrane for antibody-expressing phage enrichment and selection. Using this procedure, antibody-expressing phages against each protein were successfully isolated within two weeks. The expression profiles of the identified proteins were then acquired by immunostaining of breast tumor tissue microarrays with the antibody-expressing phages. Using this approach, expression of Eph receptor A10, TRAIL-R2 and Cytokeratin 8 in breast tumor tissues were successfully validated. These results demonstrate the antibody proteomics system is an efficient method for screening tumor-related biomarker proteins.
基于蛋白质组学的分析方法是目前用于药物开发的最有前途的生物标志物蛋白鉴定方法。然而，通过这种方法可以发现许多在病变组织中过度或低表达的候选生物标志物蛋白。因此，迫切需要建立一种有效的筛选和验证更有价值靶点的方法。在这里，我们描述了一种“抗体蛋白质组学系统”的开发，该系统利用噬菌体抗体库技术快速制备交叉反应抗体，从而促进了从众多候选物中筛选生物标志物蛋白。通过二维差异凝胶电泳分析，从乳腺癌细胞中鉴定出 16 种过表达蛋白。具体来说，从凝胶块中回收蛋白质，并对每个样品的一部分进行质谱分析。其余部分固定在硝酸纤维素膜上，用于抗体表达噬菌体的富集和选择。使用这种方法，在两周内成功地分离出了针对每种蛋白质的抗体表达噬菌体。然后，用抗体表达噬菌体对乳腺癌组织微阵列进行免疫染色，获得所鉴定蛋白的表达谱。通过这种方法，成功验证了 Eph 受体 A10、TRAIL-R2 和细胞角蛋白 8 在乳腺癌组织中的表达。这些结果表明，抗体蛋白质组学系统是一种筛选肿瘤相关生物标志物蛋白的有效方法。
