Hijazi, H.; Altoufaily, H.; Al Siblani, S.; Barbaroux, A.; Penaud, N.; Merienne, K.; Brugiere, S.; Pflieger, D.

Histone post-translational modifications (PTMs) are key regulators of gene expression, and mass spectrometry (MS) provides a powerful platform for their characterization and quantification. However, conventional histone sample preparation using derivatization of non-modified lysines by propionic anhydride (PA) presents analytical limitations, including non-detection of short hydrophilic peptides, incomplete chromatographic separation of positional isomers, and masking of endogenous lysine propionylation and butyrylation. Here, we optimized a trimethylacetic anhydride (TMA)-based derivatization workflow to improve histone PTM analysis. Compared with PA derivatization, TMA enhanced the detection of acetylated peptides, particularly short and hydrophilic species, although peptide-dependent ionization biases affected relative peptidoform quantification. We further evaluated the suitability of TMA derivatization for investigating endogenous lysine propionylation and butyrylation. Unexpectedly, we identified lysine acetyl-methylation (Kacme) at seven sites on histone H3 and validated these assignments by targeted MS using synthetic reference peptides. To investigate the metabolic dynamics of Kacme, we performed in vivo isotope tracing in mice administered 13C-labeled glucose. Unlike canonical lysine acetylation, Kacme exhibited minimal incorporation of glucose-derived acetyl groups over 2h, suggesting substantially slower turnover of its acetyl moiety. Collectively, these findings highlight the analytical advantages and quantitative limitations of TMA-based histone derivatization, while revealing Kacme as a metabolically distinct histone modification and expanding the scope of MS-based histone PTM analysis.