Nakata Katsunori, Saitoh Ryoichi, Ishigai Masaki, Imai Kazuhiro
Laboratory of Proteomics Analysis, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
Research Division, Chugai Pharmaceutical Co. Ltd, Kamakura, Kanagawa, Japan.
Biomed Chromatogr. 2018 Feb;32(2). doi: 10.1002/bmc.4063. Epub 2017 Aug 31.
Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein.
生物体中的生物学功能通常由一组相互作用的蛋白质控制，识别相互作用的蛋白质有助于理解这些功能的机制。免疫沉淀是一种利用抗体亲和力从生物样品中分离和鉴定相互作用蛋白质的方法。在本研究中，采用荧光衍生化、高效液相色谱分离和定量，最后通过高效液相色谱-串联质谱鉴定蛋白质的FD-LC-MS/MS方法，以热休克蛋白90（HSP90）作为HepaRG细胞中相互作用蛋白的模型，来鉴定免疫沉淀样品中的蛋白质。结果，分离出了已知与HSP90形成复合物的HSC70蛋白，以及三种不同类型的HSP90-β。结果表明，所提出的免疫亲和-FD-LC-MS/MS方法可用于同时检测和鉴定与特定蛋白质相互作用的蛋白质。
