A targeted proteomic approach to assess the presence of mouse-specific histone H3 variants in brain and testis

Histones are proteins in charge of DNA packaging and compaction in eukaryotic nuclei. Histone post-translational modifications (hPTM) serve as epigenetic knobs by which eukaryotic cells fine-tune DNA-templated processes such as replication, transcription and DNA damage repair. Dysregulation of hPTMs has been linked to disease states such as cancer and neurodegenerative disorders.

Classical hPTMs include acetylation and mono-, di- and tri-methylation of lysine residues. Over the past 15 years, structures have been progressively discovered that can also modify lysines: they resemble acetylation but vary in length and hydrophobicity and are collectively called acylations. The big question that has emerged is whether they play specific or only redundant roles compared to acetylation. Besides, in mouse, several sequence variants of histone H3, which differ by a handful of amino acids scattered over the sequence, have been recently described. Some specific functions have just started being attributed to a few of them.

Screening for many combinations of biologically relevant lysine acylations and methylations on several H3 variants leads to numerous isomeric peptides that are strictly isobaric. Mass spectrometry (MS) has proven crucial for identification and quantification of histones and histone PTMs. Nonetheless, data processing and interpretation of histone mass spectra is still challenging due to the absence of a decoy strategy for database search and the presence of ample isobaric peptides that require manual inspection to validate their presence.

In this study, we focused on identification and quantification of the variably modified K27-R40 stretch in canonical H3, H3.3 and two mouse-specific H3 variants (H3mm7 and H3mm13) by targeted proteomic analysis using Parallel Reaction Monitoring (PRM). To this end, we built and refined a DDA spectral library through: (1) selecting a suitable preprocessing algorithm, (2) validating PTMs by looking at informative ions in the low-mass region and (3) utilizing synthetic peptides and Indexed Retention Time (iRT) peptides to increase identification confidence. We used PRM to exclusively target variant × PTM combinations of the stretch K27-R40 of histone H3 proteins extracted from mouse testis and brain tissues. None of H3mm7 and H3mm13 variably modified H3 K27-R40 peptides were identified except the unmodified form of H3mm7. While tentatively identified in DDA analysis, these variants are easily confused with other variably modified peptidoforms from canonical H3 and H3.3, which indicates the necessity of extra measures to confirm identifications. In all, histone PTM confident characterization requires validating multiple aspects including the retention time, the presence of diagnostic ions coming from modified lysines in the low-mass range and their relative intensities, in addition to the high-mass y fragments classically considered for reading amino acid sequence.