In Vivo Quantification of Histone Acetylation Turnover and Acetyl-CoA Sources Using 2H2O Metabolic Labeling and High-Resolution Mass Spectrometry

Dysregulated histone acetylation links cellular metabolism to gene expression, but measuring its in vivo turnover remains technically challenging. Here, we introduce a 2H2O-based metabolic labeling method coupled with high-resolution Orbitrap mass spectrometry to quantify histone acetylation turnover and site occupancy in vivo. The approach leverages differential deuterium incorporation rates between fast-labeling acetyl groups and slow-labeling peptide backbones. A two-tier analytical workflow combines full-scan mass spectrometry for monoacetylated peptides with parallel reaction monitoring (PRM) for site-specific turnover and stoichiometry. Furthermore, acetyl-group 2H enrichment at the isotopic plateau provides a measure of specific substrate contributions to the acetyl-CoA pool that fuels histone acetylation. To demonstrate biological utility, we applied this approach to mice maintained on a high-carbohydrate diet or subjected to 48 h fasting to assess histone acetylation dynamics across distinct metabolic states. Acetyl-group 2H enrichment was lower during fasting, reflecting a shift toward unlabeled fatty acid-derived acetyl-CoA. Fasting accelerated acetylation turnover across multiple histone sites and selectively remodeled stoichiometry, increasing H3K23ac while decreasing H3K18ac and H4K16ac. Quantitative tracing revealed that, during fasting, fatty acid oxidation becomes an important contributor to histone acetylation, whereas glucose remains the predominant source of nucleocytosolic acetyl-CoA (supplying >60% of carbon used for acetylation). This approach enables simultaneous in vivo assessment of histone acetylation turnover, site occupancy, and acetyl-CoA substrate utilization, offering a robust platform to investigate metabolic-epigenetic crosstalk in health and disease.