Probing the specificity of protein-protein interactions by quantitative chemical proteomics

Methods Mol Biol. 2012;803:167-81. doi: 10.1007/978-1-61779-364-6_12.

Abstract

Chemical proteomics is a versatile tool to investigate protein-small molecule interactions, but can be extended to probe also secondary binding investigating small molecule-protein 1-protein 2 interactions, providing insight into protein scaffolds. This application of chemical proteomics has in particular been applied extensively to cyclic nucleotide (cAMP, cGMP) signaling. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase (PKA). Compartmentalization of PKA plays an important role in the specificity of cAMP signaling events and is mediated by interaction of the regulatory subunit (PKA-R) with A-kinase anchoring proteins (AKAPs), which often form the core of even larger protein machineries. The selective binding of AKAPs to one of the major isoforms PKA-R type I (PKA-RI) and PKA-R type II (PKA-RII) is an important feature of cAMP/PKA signaling. However, this specificity is not well established for most AKAPs. Here, we describe a chemical proteomics approach that combines cAMP-based affinity chromatography with quantitative mass spectrometry to investigate PKA-R isoform/AKAP specificity directly in lysates of cells and tissues of any origin. With this tool, several novel PKA-R/AKAP specificities can be easily resolved.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Extracts
  • Chemical Fractionation
  • Chromatography, Liquid
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • HEK293 Cells
  • Humans
  • Ion Exchange
  • Isoenzymes / metabolism
  • Isotope Labeling
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptide Hydrolases / metabolism
  • Protein Binding
  • Protein Interaction Maps*
  • Proteomics / methods*
  • Software
  • Statistics as Topic

Substances

  • Cell Extracts
  • Isoenzymes
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Peptide Hydrolases