Automated on bead derivatization and digestion for the analysis of histone acetylation dynamics

Histones are highly basic proteins that interact with DNA to form nucleosomes and belong to the most highly modified proteins. Each modification not only changes the chemical composition of the histones, but can also alters the function of the histone species. The N-terminal region of histones is rich in lysine and arginine residues, of which lysines in particular are the targets for reversible acetylation and methylation, regulating DNA interaction, chromatin state and recruitment of transcription factors and associated proteins. Histone acetylation is therefore a hallmark of epigenetics and plays an important role in cell homeostasis and the regulation of various cellular processes, including metabolism and cell growth.

Modulating histone deacetylases (HDACs) holds significant therapeutic potential for treating cancer, inflammatory, metabolic, cardiovascular, and neurological diseases. However, HDAC inhibition can cause multiple effects and toxicity due to the varied roles of histone acetylation. Therefore, selective HDAC inhibitors like benzamides are of great interest. For the characterization of the kinetic effects of HDAC inhibition on individual histone acetylation sites, we recently developed a mass spectrometric approach combining metabolic and chemical labeling (CoMetChem) generating chemically equivalent acetylated protein and histone species for quantitative comparison using in solution derivatization and digestion [1]. Employing multiple acetylation steps, each requiring manual pH adjustment per sample, to ensure quantitative conversion from lysine to acetyl lysine residues, in solution acetylation of proteins represents an elaborate and time-consuming protocol.

To overcome the limitations of in solution acetylation, we present an automatized protocol for protein acetylation and digestion using a robotic liquid handling platform for on bead derivatization with acetic anhydride followed by tryptic digestion. In combination with metabolic labelling, our automatized sample preparation workflow was applied to decipher the kinetic specificity and metabolic dependency of selective and pan- HDAC inhibitors at individual histone acetylation sites.

Acknowledgements

This work was supported by the Tyrolian Science Fund (TWF; P18903 to M.K.), the Promotion Program for Young Scientists at University of Innsbruck (P316826 to M.K.) and the Early-Stage Funding at the University of Innsbruck (P415586 to T.K.).

References

[1] A. van Pijkeren et. al, Combined Metabolic and Chemical (CoMetChem) Labeling Using Stable Isotopes-a Strategy to Reveal Site-Specific Histone Acetylation and Deacetylation Rates by LC-MS, Analytical chemistry 93 (2021) 12872–12880. https://doi.org/10.1021/acs.anal-chem.1c01359.