A Proteoform-Resolved Atlas of Human Cardiac Histones

Area of Science:

• Biochemistry
• Proteomics
• Epigenetics

Background:

• Histone variants and post-translational modifications (PTMs) are crucial for chromatin organization and gene regulation.
• Understanding the complete histone code in native human tissues is complex and challenging.

Purpose of the Study:

• To develop a comprehensive human cardiac histone proteoform atlas.
• To analyze intact histone variants with combinatorial PTMs in human myocardium.
• To establish a robust workflow for proteoform-resolved histone analysis.

Main Methods:

• A streamlined top-down proteomics workflow was employed.
• Single one-dimensional reversed-phase liquid chromatography (LC)-mass spectrometry (MS) was used for separation.
• Targeted MS/MS was utilized to resolve PTM combinations and distinguish isoforms.

Main Results:

• Baseline separation of all core histone families (H2A, H2B, H3, H4) and linker histone H1 was achieved directly from human myocardium.
• The analysis preserved the connectivity of co-occurring PTMs to individual histone molecules.
• The first comprehensive cardiac histone proteoform atlas was assembled, revealing novel histone proteoforms.

Conclusions:

• The developed top-down proteomics workflow enables proteoform-level quantification of histone composition and PTM stoichiometry.
• This approach provides a robust framework for analyzing histone variants and PTMs in complex biological systems.
• The cardiac histone proteoform atlas offers new insights into tissue-specific gene regulation.