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

Area of Science:

• Epigenetics and Molecular Biology
• Proteomics
• Biochemistry

Background:

• Histone post-translational modifications (PTMs) are crucial for gene regulation but analyzing them via mass spectrometry is hampered by complex sample preparation.
• Current workflows are time-consuming and limit the comprehensive identification and quantification of histone PTMs.

Purpose of the Study:

• To develop a streamlined, rapid, and highly efficient workflow for histone PTM analysis using mass spectrometry.
• To improve the coverage and quantitative accuracy of histone PTM identification, particularly for previously undetected modifications.

Main Methods:

• Development of RIPUP (Rapid Identification of histone PTMs in Underivatized Peptides), a multiprotease sample preparation workflow.
• Systematic evaluation of Arg-C Ultra and recombinant Chymotrypsin proteases, with and without derivatization (propionic anhydride) and TMT labeling.
• Utilized the HiP-Frag computational framework for unrestrictive PTM identification.

Main Results:

• RIPUP reduces sample preparation time to hours, enhancing PTM coverage and quantitative accuracy.
• Arg-C Ultra with TMT labeling surpassed trypsin-based methods in total PTM detection and identified a significant number of succinylation and glutarylation sites.
• The workflow successfully quantified numerous significant peptidoforms in response to nicotinamide treatment and identified diverse PTMs in rat hippocampal tissue.

Conclusions:

• RIPUP offers a rapid, high-efficiency platform for histone PTM discovery, overcoming limitations of traditional methods.
• The workflow enables the identification of a largely undetected "dark epigenome" and accelerates the discovery of epigenetic mechanisms.
• This platform facilitates the timely progression from PTM identification to therapeutic target validation in epigenetics research.