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

ABSTRACT: 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 Arg-C Ultra and a prototype recombinant (r)-Chymotrypsin proteases from Promega™ under varied conditions, with or without chemical derivatization using propionic anhydride and tandem mass tags (TMT), we demonstrate that Arg-C Ultra with TMT labeling achieves comparable total PTM detection to conventional Trypsin-based approaches. Using the HiP-Frag computational framework for unrestrictive PTM identification, we discover that TMT's tertiary amine provides charge compensation that rescues ionization of negatively charged acylation marks, revealing 50 succinylation and 27 glutarylation sites—a 'dark epigenome' largely undetected by propionylation-based methods. We demonstrate 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.

INSTRUMENT(S):

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Brain, Cell Culture

SUBMITTER: Natalie Turner  

LAB HEAD: John R Yates III

PROVIDER: PXD073683 | Pride | 2026-06-20

REPOSITORIES: Pride