Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes

Nat Protoc. 2016 Feb;11(2):316-26. doi: 10.1038/nprot.2016.020. Epub 2016 Jan 21.

Abstract

Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation-sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2-3 d from the collection of material to results.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatin / chemistry*
  • Chromatin Immunoprecipitation / methods*
  • Chromatography, Liquid / methods*
  • Mass Spectrometry / methods*
  • Proteins / analysis*
  • Time Factors

Substances

  • Chromatin
  • Proteins