title: Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners.
abstract: Affinity purification coupled to mass spectrometry (AP-MS) is an effective means of identifying protein-protein interactions to better understand biological functions. However, issues associated with sample preparation still limit the success of AP-MS for specific classes of proteins, including those associated with chromatin that exhibit overall poor solubility in the protocols normally used for AP-MS analysis. Here, we wanted to provide a generally applicable method to simultaneously identify interactors for the chromatin-bound and the soluble fractions of a given bait protein. Using four FLAG-tagged canonical histone proteins (H2A, H2B, H3.1 and H4) we demonstrate that the chromatin solubility issue can be robustly alleviated by fragmenting DNA prior to AP-MS using a combination of sonication and nuclease treatment. We show that - in comparison to a commonly used AP-MS method - our optimized protocol greatly improves the recovery of chromatin-associated interactors for core histones. Critically, this is achieved while preserving the interaction partners associated with the soluble portion of the histones. Detailed protocols amenable to the study of both histone and non-histone baits are presented here.
  
  
  CONCLUSIONS
  This manuscript describes workflow improvements to enable the recovery of chromatin-bound interactors by affinity purification coupled to mass spectrometry (AP-MS). This is significant, as most of the high-throughput studies to date can only monitor protein-protein interactions for soluble (not bound to chromatin) components. By consequence, we still poorly understand how protein complexes form on chromatin, which greatly hampers our understanding of gene expression. Using core histones as test cases, we show here a simple and universally applicable workflow that permits the identification of chromatin-bound protein-protein interactions. As exemplified in our manuscript, this revised protocol should result in a much deeper understanding of chromatin biology. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes?
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Lambert
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada.
authors.forename: Monika
authors.initials: M
authors.lastname: Tucholska
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada.
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto M5S 1A8, Canada. Electronic address: gingras@lunenfeld.ca.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
mesh_terms: D002843:Chromatin / Q000378:metabolism*; D002846:Chromatography, Affinity; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods; D012995:Solubility
chemical_list: D002843:Chromatin; D006657:Histones; D004247:DNA
keywords: Affinity purification coupled to mass spectrometry; Chromatin; Protein–protein interactions; Systems biology