Rapid Histone Post-Translational Modification Analysis Using Alternative Proteases and Tandem Mass Tags

Histone post-translational modifications (PTMs) alter chromatin dynamics and contribute to the regulation of gene expression in health and disease. Mass spectrometry-based analysis is the gold-standard for histone PTM analysis, but it remains constrained by inefficient sample preparation workflows requiring multiple days. Here, we develop RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multi-protease workflow that reduces sample preparation from days to hours while improving PTM coverage and quantitative accuracy. Through systematic evaluation of the Arg-C Ultra protease and a prototype recombinant (r)-Chymotrypsin protease under varied conditions, such as chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrated that Arg-C Ultra with TMT labeling achieves a detection of total PTM comparable to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discovered that TMTs tertiary amine provides charge compensation that rescues the ionization of negatively charged acylations revealing 50 succinylation and 27 glutarylation sites - a dark epigenome largely undetected by propionylation-based methods. We demonstrated that complementary digestion with Arg-C Ultra and r-Chymotrypsin provides orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. Application of RIPUP to frozen-thawed rat hippocampal sections within a 3-hour workflow identifies >200 PTMs including biologically critical PTM sites H3 K27/K36/K37 methylation, H4 N-terminal acetylation patterns, and H2A ubiquitination at K118/K119. This rapid, high-efficiency platform enables timely discovery of epigenetic mechanisms and accelerates the path from PTM identification to therapeutic target validation.