A Derivatization-Free Parallel Reaction Monitoring-Based Proteomics Workflow for Quantitative, Site-Specific Analysis of Histone Post-Translational Modifications.

Histone post-translational modifications (PTMs) are key regulators of chromatin architecture and gene expression. Although mass spectrometry (MS)-based data-independent acquisition (DIA) pipelines for histone PTM quantification are available, multiplexed targeted assays remain underdeveloped. Here, we present a derivatization-free parallel reaction monitoring (PRM) workflow for robust, quantitative, and site-specific analysis of major histone H3 and H4 PTMs. We employed highly efficient ArgC digestion to generate peptides of optimal length for liquid chromatography-tandem mass spectrometry (LC-MS/MS) while preserving endogenous PTMs, and we optimized chromatographic conditions to achieve isobaric separation and stable retention times. Co-eluting isobaric PTM species were confidently distinguished using site-specific fragment ions. The resulting PRM method enabled sensitive and reproducible detection of histone PTM isoforms across diverse biological systems. To illustrate its utility, we analyzed PTM dynamics in cells expressing histone H3.3 lysine-to-methionine substitutions and in cells treated with the histone deacetylase inhibitor entinostat, yielding results that correlated strongly with antibody-based readouts. This PRM platform provides a complementary, targeted approach to current chemical derivatization-based methods, enabling reliable validation of selected histone PTMs.