Histone post-translational modifications (PTMs) comprise one of the most intricate nuclear signaling networks that govern gene expression in a long-term and dynamic fashion. These PTMs are considered to be ‘epigenetic’ or heritable from one cell generation to the next and help establish genomic expression patterns. While much of the analyses of histones have historically been performed using site-specific antibodies, these methods are replete with technical obstacles (i.e., cross-reactivity and epitope occlusion). Mass spectrometry-based proteomics has begun to play a significant role in the interrogation of histone PTMs, revealing many new aspects of these modifications that cannot be easily determined with standard biological approaches. Here, we review the accomplishments of mass spectrometry in the histone field, and outline the future roadblocks that must be overcome for mass spectrometry-based proteomics to become the method of choice for chromatin biologists.

X chromosome dosage compensation is required in male Drosophila to increase gene expression from the single X to equal that of both female X chromosomes. Although this is a sex-specific process, the MSL dosage compensation complex is thought to interface with non-sex-specific factors for both targeting and function. Therefore, a biochemical approach to MSL-associated factors is needed to complement genetic studies based on male-specific lethality. Here, we applied chromatin interacting protein-mass spectrometry (ChIP-MS) to identify MSL interactions on crosslinked chromatin, rather than focusing solely on complexes released from the DNA. Using this approach we identified MSL-enriched histone modifications, CG1832, a zinc finger protein implicated in initial MSL localization, and CG4747, a putative H3K36me3 binding protein. We found that CG4747 is associated with the bodies of active genes, coincident with H3K36me3, and is mis-localized in the set2 mutant lacking H3K36me3. CG4747 loss-of-function in vivo results in partial mis-localization of MSL complex to autosomes, and RNAi in cell culture confirms that CG4747 and SET2 function together to facilitate targeting of MSL complex to active genes. Our results demonstrate that the combination of crosslinking, affinity-purification, and mass spectrometry is a promising avenue for discovery of functional interactions on the chromatin template.

The connections between various nuclear processes and specific histone posttranslational modifications are dependent to a large extent on the acquisition of those modifications after histone synthesis. The reestablishment of histone posttranslational modifications after S phase is especially critical for H3K9 and H3K27 trimethylation, both of which are linked with epigenetic memory and must be stably transmitted from one cellular generation to the next. This report uses a proteomic strategy to interrogate how and when the cell coordinates the formation of histone posttranslational modifications during division. Paramount among the findings is that H3K9 and H3K27 trimethylation begins during S phase but is completed only during the subsequent G 1 phase via two distinct pathways from the unmodified and preexisting dimethylated states. In short, we have systematically characterized the temporal origins and methylation pathways for histone posttranslational modifications during the cell cycle.

Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

Mass spectrometry (MS)-based proteomics has become the most utilized tool to characterize histone post-translational modifications (PTMs). Since histones are highly enriched in lysine and arginine residues, lysine derivatization has been developed to prevent the generation of short peptides (3-6 residues) during trypsin digestion. One of the most adopted protocols applies propionic anhydride for derivatization. However, the propionyl group is not sufficiently hydrophobic to fully retain the shortest histone peptides in reversed-phase liquid chromatography, and such procedure also hampers the discovery of natural propionylation events. In this work we tested 12 commercially available anhydrides, selected based on their safety and hydrophobicity. Performance was evaluated in terms of yield of the reaction, MS/MS fragmentation efficiency and drift in retention time by using the following samples: (i) a synthetic unmodified histone H3 tail, (ii) synthetic modified histone peptides and (iii) a histone extract from cell lysate. Results highlighted that 7 of the selected anhydrides increased peptide retention time as compared to propionic, and several anhydrides such as benzoic and valeric led to high MS/MS spectra quality. However, propionic anhydride derivatization still resulted in our opinion as the best protocol to achieve high MS sensitivity and more even ionization efficiency among the analyzed peptides.