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

Hisham Mohammed, Christopher Taylor, Gordon D. Brown, Evaggelia K. Papachristou, Jason S. Carroll, Clive S. D'Santos

Research output: Contribution to journalReview articlepeer-review

84 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)316-326
Number of pages11
JournalNature protocols
Volume11
Issue number2
DOIs
StatePublished - Feb 1 2016
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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