BackgroundThe concept that individual traits can be acquired and transmitted by the germline through epigenetic mechanisms has gained recognition in the past years. However, epigenetic marks in sperm have not been are not well identified.ResultsUsing a novel proteomic approach that combines peptide-based bottom-up and intact protein top-down tandem mass spectrometry, we report the identification of epigenetic marks on histones and protamines in adult mouse sperm. We identified a total of 26 post-translational modifications (PTMs) on specific residues of the core histones H2B, H3 and H4, and the linker histone H1, four of which had not been described previously in any tissue or cell line. We also detected 11 novel PTMs on the protamines PRM1 and PRM2 and observed that they are present in specific combinations on individual protamines.ConclusionsBoth histones and protamines carry multiple PTMs in the adult mouse sperm. On protamines, specific PTM combinations might form a ‘protamine code’ similar to the ‘histone code’. These findings suggest a potential role for PTMs on sperm histones and protamines in epigenetic signatures underlying transgenerational inheritance.

Post-translational modifications (PTMs) of proteins are biochemical processes required for cellular functions and signalling that occur in every sub-cellular compartment. Multiple protein PTMs exist, and are established by specific enzymes that can act in basal conditions and upon cellular activity. In the nucleus, histone proteins are subjected to numerous PTMs that together form a histone code that contributes to regulate transcriptional activity and gene expression. Despite their importance however, histone PTMs have remained poorly characterised in most tissues, in particular the brain where they are thought to be required for complex functions such as learning and memory formation. Here, we report the comprehensive identification of histone PTMs, of their combinatorial patterns, and of the rules that govern these patterns in the adult mouse brain. Based on liquid chromatography, electron transfer, and collision-induced dissociation mass spectrometry, we generated a dataset containing a total of 10,646 peptides from H1, H2A, H2B, H3, H4, and variants in the adult brain. 1475 of these peptides carried one or more PTMs, including 141 unique sites and a total of 58 novel sites not described before. We observed that these PTMs are not only classical modifications such as serine/threonine (Ser/Thr) phosphorylation, lysine (Lys) acetylation, and Lys/arginine (Arg) methylation, but also include several atypical modifications such as Ser/Thr acetylation, and Lys butyrylation, crotonylation, and propionylation. Using synthetic peptides, we validated the presence of these atypical novel PTMs in the mouse brain. The application of data-mining algorithms further revealed that histone PTMs occur in specific combinations with different ratios. Overall, the present data newly identify a specific histone code in the mouse brain and reveal its level of complexity, suggesting its potential relevance for higher-order brain functions.