A Derivatization-Free Parallel Reaction Monitoring-Based Proteomics Workflow for Quantitative, Site-Specific Analysis...

Area of Science:

• Biochemistry
• Molecular Biology
• Epigenetics

Background:

• Histone post-translational modifications (PTMs) are crucial for regulating chromatin structure and gene expression.
• Existing mass spectrometry (MS) methods for quantifying histone PTMs, particularly data-independent acquisition (DIA), are available, but multiplexed targeted assays are less developed.

Purpose of the Study:

• To present a derivatization-free parallel reaction monitoring (PRM) workflow for quantitative, site-specific analysis of major histone H3 and H4 PTMs.
• To establish a robust and sensitive method for detecting histone PTM isoforms across various biological systems.

Main Methods:

• Utilized ArgC digestion for optimal peptide generation for liquid chromatography-tandem mass spectrometry (LC-MS/MS).
• Optimized chromatographic conditions for isobaric separation and stable retention times.
• Employed site-specific fragment ions to distinguish co-eluting isobaric PTM species.

Main Results:

• Developed a sensitive and reproducible PRM method for histone PTM analysis.
• Demonstrated the method's utility in analyzing PTM dynamics in cells with histone H3.3 substitutions and entinostat treatment.
• Achieved strong correlation of results with antibody-based readouts.

Conclusions:

• The PRM platform provides a reliable, targeted alternative to chemical derivatization-based methods.
• Enables accurate validation of specific histone PTMs.
• Facilitates deeper understanding of epigenetic regulation and gene expression.