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

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

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.

MeSH terms

  • Cell Line
  • Chromatography, Affinity / methods*
  • Chromatography, High Pressure Liquid / methods
  • HSP90 Heat-Shock Proteins / analysis
  • HSP90 Heat-Shock Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • Protein Binding
  • Proteins / analysis*
  • Proteins / metabolism*
  • Proteomics / methods*
  • Tandem Mass Spectrometry / methods*

Substances

  • HSP90 Heat-Shock Proteins
  • Proteins