Mass spectrometry (MS)-based characterization is important in proteomic research for verification of structural features and functional understanding of gene expression. Post-translational modifications (PTMs) such as methylation and acetylation have been reported to be associated with chromatin remodeling during spermatogenesis. Although antibody- and MS-based approaches have been applied for characterization of PTMs on H3 variants during spermatogenesis, variant-specific PTMs are still underexplored. We identified several lysine modifications in H3 variants, including testis-specific histone H3 (H3t), through their successful separation with MS-based strategy, based on differences in masses, retention times, and presence of immonium ions. Besides methylation and acetylation, we detected formylation as a novel PTM on H3 variants in mouse testes. These patterns were also observed in H3t. Our data provide high-throughput structural information about PTMs on H3 variants in mouse testes and show possible applications of this strategy in future proteomic studies on histone PTMs.
Keywords: DTT, dithiothreitol; ESI-TRAP, electrospray TRAP; FDR, false discovery rate; H2SO4, sulfuric acid; HCD, high-energy collision dissociation; HFBA, heptafluorobutyric acid; HPLC, high performance liquid chromatography; ISD, in source decay; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; Mass spectrometry; PTMs, post-translational modifications; Post-translational modification; RP, reverse phase; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Spermatogenesis; TCA, trichloroacetic acid; TFA, trifluoroacetic acid; Testis-specific H3 histone.