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Improving the recovery of chromatin-bound protein complexes via chromatin solubilisation in affinity purification coupled to mass spectrometry
First Author: Jean-Philippe Lambert
Abstract:
  Affinity purification coupled to mass spectrometry (AP-MS) is an effective mean 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. Here we report an integrated proteomic workflow specifically designed to characterize mammalian protein complexes associated with chromatin. We demonstrate that a major hurdle facing the purification of chromatin-associated protein complexes is their low solubility and that this issue can be alleviated by fragmenting chromatin prior to AP-MS. Briefly, four canonical histone proteins (H2A, H2B, H3.1 and H4) were N-terminally tagged with 3XFLAG, expressed stably in HEK293 cells, and analyzed by AP-MS using either a previously published protocol (Kean et al., Methods, 2012) or a revised AP-MS method optimized for chromatin-associated proteins. Here we demonstrate that our optimized protocol greatly improves the recovery of chromatin-associated core histone interactors without losing soluble interaction partners identified by our published protocol. In this report, we present the details of our complete proteomic workflow solution for the expression, purification and analysis of chromatin-associated protein complexes. This protocol is amenable to the study of both histone and non-histone baits.


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