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

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.

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Figure 1
Figure 2: Total ion current, full chromatogram of ERα-RIME.
Figure 3: Example of RIME experiments for ERα and E2F1.
Figure 4: Amino acid sequence of ERα.

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Acknowledgements

We acknowledge the support of the University of Cambridge and Cancer Research UK. J.S.C. is supported by a European Research Council (ERC) starting grant and a European Molecular Biology Organization (EMBO) Young investigator award.

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Contributions

H.M., J.S.C. and C.S.D. designed the research. H.M., C.T., E.P., G.D.B., J.S.C. and C.S.D. performed the experiments and analyzed the data. H.M. and C.S.D. wrote the manuscript. H.M., C.T., E.P., G.D.B., J.S.C. and C.S.D. edited the manuscript.

Corresponding authors

Correspondence to Hisham Mohammed or Clive S D'Santos.

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Mohammed, H., Taylor, C., Brown, G. et al. Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) for analysis of chromatin complexes. Nat Protoc 11, 316–326 (2016). https://doi.org/10.1038/nprot.2016.020

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