Systematic quantitative analysis of H2A and H2B variants by targeted proteomics

Area of Science:

• Molecular Biology
• Proteomics
• Epigenetics

Background:

• Histone variants are crucial for DNA organization into chromatin and functional modulation.
• Traditional antibody-based assays struggle to differentiate between highly similar histone variants.
• There is a need for precise methods to quantify histone variants and their isoforms.

Purpose of the Study:

• To establish a mass spectrometry-based targeted proteomics method for quantifying numerous histone variants simultaneously.
• To overcome the limitations of antibody-based assays in distinguishing closely related histone variants.
• To apply this method to analyze histone variant expression in complex biological samples.

Main Methods:

• Developed a targeted proteomics assay using selected reaction monitoring (SRM) or parallel reaction monitoring (PRM).
• Utilized 55 peptides representing 25 distinct histone sequences, including those differing by a single amino acid.
• Applied the method to crude extracts, specifically analyzing H2A and H2B variants in mouse testis.

Main Results:

• Successfully quantified a maximum number of histone variants in a single multiplexed assay.
• Confirmed abundance profiles of testis-specific histones during spermatogenesis.
• Identified predicted H2A.L.1 isoforms and explored their overexpression in a male infertility model.

Conclusions:

• Targeted proteomics is a powerful tool for quantifying highly similar histone variants and isoforms.
• The developed method can be readily adapted for studying human histone variants.
• This approach has potential applications in understanding diseases associated with deregulated histone variant abundance.