Investigating Histone Lysine Acetylation and Acetyl-methylation in Mouse Brain using Enhanced Trimethylacetic Anhydride Derivatization Protocol

Researchers have developed an enhanced method for studying histone post-translational modifications (PTMs) in the mouse brain using mass spectrometry (MS). Traditional histone sample preparation often fails to detect short, hydrophilic peptides, and fails to fully separate positional isomers or mask endogenous modifications like lysine propionylation and butyrylation.

To overcome these limitations, the researchers optimized a trimethylacetic anhydride (TMA)-based derivatization protocol. This new approach improved the detection of acetylated peptides, particularly those that are short and hydrophilic, although some ionization biases remained peptide-dependent.

While TMA derivatization enhanced the detection of acetylation, it also revealed a previously unknown modification: lysine acetyl-methylation (Kacme) at seven sites on histone H3. This modification was validated through targeted MS using synthetic reference peptides.

In an in vivo study, the researchers administered 13C-labeled glucose to mice and monitored the metabolic dynamics of Kacme. Unlike standard lysine acetylation, Kacme did not incorporate glucose-derived acetyl groups over a 2-hour period, suggesting a significantly slower turnover rate for its acetyl moiety.

Overall, this study demonstrates the benefits and limitations of TMA-based histone derivatization, while also identifying Kacme as a distinct and metabolically unique histone modification, thereby expanding the possibilities for mass spectrometry-based analysis of histone PTMs.