The interaction of Oct4 and the nucleosome remodeling deacetylase (NuRD) HDAC complex member SALL4 stresses its role in modulating epigenetic silencing [92]. Joshi et al. probes able to decipher small molecule targets and off-targets in a close-to-native environment. These are small molecule analogues of epigenetic drugs conceived as protein target enrichment tools after they have engaged them in cells or lysates. Such probes, which have been designed NS6180 for deacetylases, bromodomains, demethylases, and methyltransferases not only enrich their direct protein targets but also their stable interactors, which can be identified by mass spectrometry. Hence, they constitute a tool to study the epigenetic complexes together with other techniques also reviewed here: immunoaffinity purification with antibodies against native protein complex constituents or epitope tags, affinity matrices designed to bind recombinantly tagged protein, and enrichment of the complexes using histone tail peptides as baits. We expect that this toolbox will be adopted by more and more researchers willing to harness the spectacular advances in mass spectrometry to the epigenetic field. Keywords: Target deconvolution, Complex identification, Affinity matrix, Photo-cross-linking, Affinity purification, Immunoaffinity purification Background Proteomics has proven to be a reliable ally to study drug-protein interactions and protein-protein interactions for the last 20?years [1]. Substantial progress in the mass spectrometry techniques now allows to measure accurately and quantitatively the proteins contained in complex samples. Advances in the technology allow shorter measurement times while digging deeper in the proteomes. Hydrolases and kinases have been the great beneficiaries of past efforts to characterize sub-proteomes and their inhibitors [2, 3]. But they are not the only ones. The field of epigenetic proteomics could develop further as it accompanies the thriving phase of epigenetic drug discovery endeavors and successes [4C6] outlined throughout this special issue. Chemical proteomics methods to identify epigenetic drug targets and off-targets Among the contributions made by proteomics to the field of epigenetics [5], chemical proteomics [7] has emerged as a solid methodology to decipher small molecule targets and off-targets via so-called compound pulldowns. These consist of the enrichment of the sub-proteome that is bound by a small molecule in a complex lysate or even directly in cells and generally mass spectrometry readout. Because of unspecific binding or cross-linking, the sub-proteome enriched by inactive molecules can be compared as a control. However, a better strategy is to compete with the free small molecule, preferably in a dose-dependent fashion, where the fit of the curves will help determine the reality of the newly found targets. Additionally, such NS6180 a competition assay will give EC50s, which can be converted to binding that is addressable by a molecule equipped with a handle allowing post-lysis pulldowns. In this case, a cross-linker can also be added. It has to be noted that any modification of the initial molecule can impair binding, that the bulk and length of the linker matters, and that cross-linking can be relatively low-yielding and unspecific [15]. Hence, we propose to distinguish (Fig.?1) between the: Small molecule ligand immobilized on a solid matrix. Different solid matrices can be envisioned, the most common being Sepharose beads or magnetic beads. Small molecule ligand functionalized with an enrichment handle. This enrichment handle can be a biotin NS6180 moiety, allowing subsequent enrichment with a streptavidin matrix. It can also be a biorthogonal tag allowing for further enrichment using click reactions [16, 17]. Small molecule ligand functionalized with a cross-linking group and an enrichment handle. The cross-linking group are very often photoreactive functionalities such as benzophenones, aryl azides, or diazirines [18, 19]. Open in a separate window Fig. 1 Affinity probes for the identification of drug targets by chemical proteomics strategies. An analogue of the small molecule is synthesized that a is covalently attached to a solid matrix or b possesses an enrichment handle or c possesses a cross-linking moiety and an enrichment handle Deacetylase enrichment probes Since the HDAC inhibitor Vorinostat (aka SAHA) has been the epigenetic drug the most studied by chemical proteomics, we shall begin by describing the various reported approaches using linkable analogues of this molecule. They constitute a good overview of what is possible in the field: all the approaches described above (Fig.?1) ER81 have indeed been successfully used for the identification of Vorinostat targets. A team of researchers in Cellzome immobilized a linkable analogue of Vorinostat (p-aminomethyl Vorinostat) and an analogue of Givinostat on Sepharose beads to obtain an affinity matrix able to enrich HDAC1, 2, 3, 6, 8, and 10 out.