RIPUP workflow rapidly identifies 58 succinylation and 31 glutarylation sites, improving histone PTM analysis
Background
Histone post-translational modifications (PTMs) are crucial regulators of gene expression and chromatin dynamics, playing significant roles in both health and disease. Current mass spectrometry-based methods for analyzing histone PTMs are often hampered by inefficient sample preparation workflows, limiting their throughput and comprehensive coverage. This inefficiency creates a bottleneck in rapidly identifying novel epigenetic mechanisms and validating potential therapeutic targets, particularly for less common or negatively charged acylations that are difficult to detect.
Study Design
Researchers developed RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a streamlined multiprotease workflow designed to accelerate histone PTM analysis. They systematically evaluated alternative proteases, Arg-C Ultra and recombinant (r)-Chymotrypsin, under various conditions, including standard derivatization with propionic anhydride and tandem mass tag (TMT) labeling. The RIPUP workflow was applied to HEK293T cells treated with the pan-sirtuin inhibitor nicotinamide at varying doses, and also to frozen-thawed rat hippocampal sections. PTM identification utilized the HiP-Frag computational framework.
Results
The RIPUP workflow significantly reduced sample preparation time to just hours while enhancing PTM coverage and quantitative accuracy. > Arg-C Ultra with TMT labeling achieved superior total PTM detection compared to traditional Trypsin-based approaches. The study revealed that TMT's tertiary amine provides crucial charge compensation, rescuing the ionization of negatively charged acylations. This led to the discovery of 58 succinylation and 31 glutarylation sites, termed a "dark epigenome," which were largely undetected by propionylation-based methods. Complementary digestion with Arg-C Ultra and r-Chymotrypsin provided orthogonal sequence coverage, enabling detection of PTMs in H2A variants, linker histones, and regions poorly represented by arginine-specific cleavage alone. In HEK293T cells, RIPUP quantified 112 statistically significant peptidoforms (adj p < 0.05) following nicotinamide treatment, with 88 increasing and 24 decreasing in a dose-dependent manner. Application to rat hippocampal sections within a 3 h workflow identified >200 PTMs, including H3 K27/K36/K37 methylation, H4 N-terminal acetylation, and H2A K118/K119 ubiquitination.
Why It Matters
This RIPUP platform represents a significant leap forward for epigenetic research and drug discovery, drastically cutting the time required for histone PTM analysis. Faster, more comprehensive PTM mapping will accelerate the identification of novel epigenetic mechanisms implicated in disease, moving from PTM identification to therapeutic target validation more efficiently. This method could enable high-throughput screening for compounds that modulate specific PTMs, potentially uncovering new drug targets or optimizing existing therapies. For researchers, this means quicker insights into cellular processes and disease progression, potentially leading to more rapid development of interventions that target specific epigenetic pathways.