A proteomics method using immunoaffinity fluorogenic derivatization–liquid chromatography/tandem mass spectrometry (FD‐LC‐MS/MS) to identify a set of interacting proteins

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EarlyView Article

  • Published: Aug 31, 2017
  • Author: Katsunori Nakata, Ryoichi Saitoh, Masaki Ishigai, Kazuhiro Imai
  • Journal: Biomedical Chromatography

Abstract

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.

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