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

• Published in: Nature protocols Vol. 11; no. 2; pp. 316 - 326
• Main Authors: Mohammed, Hisham, Taylor, Christopher, Brown, Gordon D, Papachristou, Evaggelia K, Carroll, Jason S, D'Santos, Clive S
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.02.2016; Nature Publishing Group
• Subjects: 631/1647/296; 631/1647/664/2089; 631/45/475; 631/45/612/100; Amino acids; Analytical Chemistry; Antibodies; Biological Techniques; Cellular proteins; Chromatin; Chromatin - chemistry; Chromatin Immunoprecipitation - methods; Chromatography, Liquid - methods; Computational Biology/Bioinformatics; Contamination; Formaldehyde; Genetic research; Genetic transcription; Immunoprecipitation; Kinases; Life Sciences; Ligands; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Methods; Microarrays; Organic Chemistry; Peptides; Properties; Protein purification; Proteins; Proteins - analysis; Proteomics; Protocol; Rime; Scientific imaging; Spectroscopy; Time Factors; Transcription factors; Yeast; United Kingdom > UK
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2016.020

This protocol describes affinity purification of endogenous protein complexes for mass spectrometry analysis. Optimized to study formaldehyde-crosslinked proteins isolated by chromatin immunoprecipitation, it can be adapted to study other protein complexes. 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.