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

Area of Science:

• Epigenetics and Molecular Biology
• Proteomics and Mass Spectrometry
• Chromatin Biology

Background:

• Histone post-translational modifications (PTMs) are crucial for gene regulation.
• Current mass spectrometry workflows for histone PTM analysis are time-consuming, often taking days.
• There is a need for faster, more efficient methods to study histone PTMs.

Purpose of the Study:

• To develop a streamlined, rapid sample preparation workflow for histone PTM identification.
• To improve the coverage and quantitative accuracy of histone PTM analysis.
• To uncover previously undetected histone PTMs and expand the understanding of the 'dark epigenome'.

Main Methods:

• Development of RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a multi-protease digestion workflow.
• Systematic evaluation of Arg-C Ultra and recombinant Chymotrypsin proteases.
• Application of tandem mass tags (TMT) for enhanced PTM detection and propionic anhydride derivatization for comparison.
• Utilizing the HiP-Frag computational framework for PTM identification.
• Complementary digestion strategies for orthogonal sequence coverage.

Main Results:

• RIPUP reduces sample preparation time from days to hours.
• TMT labeling with Arg-C Ultra achieves PTM detection comparable to conventional methods.
• TMT labeling rescues ionization of negatively charged acylations, revealing numerous succinylation and glutarylation sites.
• Complementary digestion enables PTM detection in challenging histone regions and variants.
• Over 200 PTMs, including critical methylation, acetylation, and ubiquitination sites, were identified in rat hippocampal sections within a 3-hour workflow.

Conclusions:

• RIPUP is a rapid, high-efficiency platform for histone PTM discovery.
• The method accelerates the identification of epigenetic regulatory mechanisms.
• RIPUP facilitates faster validation of PTMs as therapeutic targets.