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. 2014 Apr 4:100:55-9.
doi: 10.1016/j.jprot.2013.12.022. Epub 2014 Jan 9.

Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners

Jean-Philippe Lambert  1 , Monika Tucholska  1 , Tony Pawson  2 , Anne-Claude Gingras  3
Affiliations

Incorporating DNA shearing in standard affinity purification allows simultaneous identification of both soluble and chromatin-bound interaction partners

Jean-Philippe Lambert et al. J Proteomics. .

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.

Biological significance: 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?

Keywords: Affinity purification coupled to mass spectrometry; Chromatin; Protein–protein interactions; Systems biology.

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Figures

Figure 1
Figure 1
DNA shearing facilitates the affinity purification of chromatin-associated proteins. (A) Phenol/chloroform extracted DNA from passively lysed cells, lysed cells which were further sonicated or both sonicated and treated with benzonase, was resolved on a 1% agarose gel and stained with ethidium bromide. Note that DNA from passively lysed cells did not enter the agarose gel due to its large size. (B) Chromatin shearing increases the solubility of 3XFLAG tagged histone baits. Cell extracts and affinity purified samples from HEK293 cells stably expressing 3XFLAG histones or an empty control vector were resolved on a 4-15% Criterion SDS-PAGE gels and immunoblotted with antibodies as indicated. (C) Chromatin shearing increases the solubility of chromatin-associated proteins and their recovery as histone interactors.
Figure 2
Figure 2
The use of DNA shearing in AP sample preparation enhances the identification by mass spectrometry of chromatin-bound interactors for core histone proteins. A heat map of the interaction partners identified with each histone bait using the “standard” or chromatin optimized AP approach (Avg SAINT ≥ 0.8) was generated using MeV 4.81. The pixel color corresponds to the total number of peptides (spectral counts) detected for a given prey across all replicates. Interactor localization and functions, as defined by GO annotation, is highlighted below the heat map.
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