Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition

Nat Methods. 2013 Dec;10(12):1239-45. doi: 10.1038/nmeth.2702. Epub 2013 Oct 27.

Abstract

Characterizing changes in protein-protein interactions associated with sequence variants (e.g., disease-associated mutations or splice forms) or following exposure to drugs, growth factors or hormones is critical to understanding how protein complexes are built, localized and regulated. Affinity purification (AP) coupled with mass spectrometry permits the analysis of protein interactions under near-physiological conditions, yet monitoring interaction changes requires the development of a robust and sensitive quantitative approach, especially for large-scale studies in which cost and time are major considerations. We have coupled AP to data-independent mass spectrometric acquisition (sequential window acquisition of all theoretical spectra, SWATH) and implemented an automated data extraction and statistical analysis pipeline to score modulated interactions. We used AP-SWATH to characterize changes in protein-protein interactions imparted by the HSP90 inhibitor NVP-AUY922 or melanoma-associated mutations in the human kinase CDK4. We show that AP-SWATH is a robust label-free approach to characterize such changes and propose a scalable pipeline for systems biology studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Chromatography, Affinity / methods*
  • Chromatography, Liquid / methods
  • Cyclin-Dependent Kinase 4 / chemistry
  • Cyclin-Dependent Kinase 4 / genetics
  • Gene Library
  • Humans
  • Isoxazoles / chemistry
  • Mass Spectrometry / methods*
  • Mutation
  • Principal Component Analysis
  • Protein Interaction Mapping / methods*
  • Proteins / chemistry
  • Resorcinols / chemistry
  • Systems Biology

Substances

  • 5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid ethylamide
  • Isoxazoles
  • Proteins
  • Resorcinols
  • CDK4 protein, human
  • Cyclin-Dependent Kinase 4