title: 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.
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Hisham
authors.initials: H
authors.lastname: Mohammed
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: Taylor
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Gordon D
authors.identifier: http://orcid.org/0000-0003-4922-936X
authors.initials: GD
authors.lastname: Brown
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Evaggelia K
authors.initials: EK
authors.lastname: Papachristou
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Jason S
authors.initials: JS
authors.lastname: Carroll
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK.
authors.forename: Clive S
authors.identifier: http://orcid.org/0000-0003-4592-0487
authors.initials: CS
authors.lastname: D'Santos
mesh_terms: D002843:Chromatin / Q000737:chemistry*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D013997:Time Factors
chemical_list: D002843:Chromatin; D011506:Proteins