Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.
Immuno-proteomics: Development of a novel reagent for separating antibodies from their target proteins.
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免疫蛋白质组学：开发一种用于将抗体与其靶蛋白分离的新型试剂。
DOI：
10.1016/j.bbapap.2014.10.011
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Minden,Jonathan
中科院分区：
文献类型：
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作者：
Ganesan,Vinitha;Schmidt,Brigitte;Avula,Raghunandan;Cooke,Dagney;Maggiacomo,Taylor;Tellin,Lawton;Ascherman,DanaP;Bruchez,MarcelP;Minden,Jonathan
免疫沉淀（IP）技术是一种广泛应用于鉴定特异性抗体靶蛋白结合伙伴的技术。假定的结合靶标及其伴侣的数量通常比用于捕获这些靶蛋白的抗体要少得多。因此，利用蛋白质组学进行抗原鉴定往往会被大量干扰性抗体蛋白的存在所混淆。即使是共价连接的抗体珠在IP期间也容易被抗体浸出。为了避免这种干扰，我们在这里描述了一种试剂，称为生物素- cdm，它可以用生物素可逆地标记细胞裂解液中所有潜在的靶蛋白。与靶蛋白偶联的生物素的存在允许二级分离步骤，其中抗体通过将其与亲和素偶联的基质结合而从可逆生物素化的靶蛋白中洗掉。捕获的目标蛋白通过逆转生物素- cdm链接从亲和素基质中释放出来，从而释放出一组目标蛋白，准备进行与2d电泳兼容的进一步蛋白质组学分析。在这里，我们描述了生物素- cdm的合成和表征。我们还证明了生物素- cdm用于已知抗原的免疫沉淀，以及用于捕获一系列由类风湿关节炎患者血清中发现的自身抗体靶向的蛋白质。使用该试剂可以将免疫沉淀和2d差异凝胶电泳结合起来，克服了目前血清学蛋白质组分析（SERPA）在发现自身抗原方面的局限性。本文是《医学蛋白质组学》特刊的一部分。
Immunoprecipitation (IP) is a widely used technique for identifying the binding partners of the target proteins of specific antibodies. Putative binding targets and their partners are usually in much lower amounts than the antibodies used to capture these target proteins. Thus antigen identification using proteomics following IP is often confounded by the presence of an overwhelming amount of interfering antibody protein. Even covalently linking antibodies to beads is susceptible to antibody leaching during IP. To circumvent this interference, we describe here a reagent, called Biotin-CDM that reversibly tags all potential target proteins in a cell lysate with biotin. The presence of biotin coupled to the target proteins allows for a secondary separation step in which antibodies are washed away from the reversibly biotinylated target proteins by binding them to an Avidin-coupled matrix. The captured target proteins are released from the Avidin matrix by reversing the Biotin-CDM link, thus releasing a pool of target proteins ready for further proteomic analysis compatible with 2D-electrophoresis. Here, we describe the synthesis and characterization of Biotin-CDM. We also demonstrate Biotin-CDM's use for immunoprecipitation of a known antigen, as well as its use for capturing an array of proteins targeted by the autoantibodies found in the serum a patient suffering from rheumatoid arthritis. The use of this reagent allows one to combine immunoprecipitation and 2D-Difference gel electrophoresis, overcoming the current limitations of Serological Proteome Analysis (SERPA) in discovering autoantigens. This article is part of a Special Issue entitled: Medical Proteomics.