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Biomed Chromatogr. 2018 Feb;32(2). doi: 10.1002/bmc.4063. Epub 2017 Aug 31.

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

Author information

1
Laboratory of Proteomics Analysis, Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, Japan.
2
Research Division, Chugai Pharmaceutical Co. Ltd, Kamakura, Kanagawa, Japan.

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.

KEYWORDS:

DAABD-Cl; FD-LC-MS/MS method; HSP90; HepaRG; immunoprecipitation

PMID:
28801948
DOI:
10.1002/bmc.4063
[Indexed for MEDLINE]

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