Mallikaratchy Prabodhika, Tang Zhiwen, Kwame Sefah, Meng Ling, Shangguan Dihua, Tan Weihong
Department of Chemistry, Shands Cancer Center, University of Florida Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611, USA.
Mol Cell Proteomics. 2007 Dec;6(12):2230-8. doi: 10.1074/mcp.M700026-MCP200. Epub 2007 Sep 17.
The identification of tumor related cell membrane protein targets is important in understanding tumor progression, the development of new diagnostic tools, and potentially for identifying new therapeutic targets. Here we present a novel strategy for identifying proteins that are altered in their expression levels in a diseased cell using cell specific aptamers. Using an intact viable B-cell Burkitt's lymphoma cell line (Ramos cells) as the target, we have selected aptamers that recognize cell membrane proteins with high affinity. Among the selected aptamers that showed different recognition patterns with different cell lines of leukemia, the aptamer TD05 showed binding with Ramos cells. By chemically modifying TD05 to covalently cross-link with its target on Ramos cells to capture and to enrich the target receptors using streptavidin coated magnetic beads followed by mass spectrometry, we were able to identify membrane bound immunoglobin heavy mu chain as the target for TD05 aptamer. Immunoglobin heavy mu chain is a major component of the B-cell antigen receptor, which is expressed in Burkitt's lymphoma cells. This study demonstrates that this two step strategy, the development of high quality aptamer probes and then the identification of their target proteins, can be used to discover new disease related potential markers and thus enhance tumor diagnosis and therapy. The aptamer based strategy will enable effective molecular elucidation of disease related biomarkers and other interesting molecules.
肿瘤相关细胞膜蛋白靶点的鉴定对于理解肿瘤进展、开发新的诊断工具以及潜在地识别新的治疗靶点都很重要。在此，我们提出了一种新策略，利用细胞特异性适配体来鉴定在病变细胞中表达水平发生改变的蛋白质。以完整存活的B细胞伯基特淋巴瘤细胞系（拉莫斯细胞）为靶点，我们筛选出了能高亲和力识别细胞膜蛋白的适配体。在筛选出的对不同白血病细胞系表现出不同识别模式的适配体中，适配体TD05显示出与拉莫斯细胞结合。通过对TD05进行化学修饰，使其与拉莫斯细胞上的靶点共价交联，然后用链霉亲和素包被的磁珠捕获并富集靶点受体，随后进行质谱分析，我们能够鉴定出膜结合免疫球蛋白重链μ链是TD05适配体的靶点。免疫球蛋白重链μ链是B细胞抗原受体的主要成分，在伯基特淋巴瘤细胞中表达。这项研究表明，这种两步策略，即开发高质量的适配体探针，然后鉴定其靶蛋白，可用于发现新的疾病相关潜在标志物，从而加强肿瘤诊断和治疗。基于适配体的策略将能够有效地对疾病相关生物标志物和其他有趣分子进行分子解析。
