One-Pot Quantitative Top- and Middle-Down Analysis of GluC-Digested Histone H4

Area of Science:

• Proteomics
• Epigenetics
• Mass Spectrometry

Background:

• Histone post-translational modifications (PTMs) are crucial for eukaryotic genome regulation.
• Current proteomics methods often fail to capture single-molecule PTM combinations (proteoforms).
• Middle-down and top-down mass spectrometry (MS) offer potential for H4 proteoform analysis.

Purpose of the Study:

• To demonstrate the effectiveness of GluC-based middle-down MS for H4 proteoform quantitation.
• To compare GluC-based middle-down MS with top-down MS for H4 proteoform analysis.
• To evaluate the concordance, biases, and complementarity of these approaches.

Main Methods:

• Utilized GluC digestion for middle-down MS analysis of histone H4.
• Compared GluC-based middle-down MS with top-down MS quantitation.
• Analyzed chromatographically resolved proteoforms from intact H4 and GluC digestion products (E52, E53, E63) in a single LC-MS run.

Main Results:

• Successfully analyzed H4 proteoforms using GluC-based middle-down MS, overcoming previous limitations.
• Observed chromatographic resolution of proteoforms from intact H4 and all three GluC products.
• Found moderate proteoform discordance between different GluC digestion products but high concordance within each product.
• Demonstrated comparable quantitation capacity between top-down and middle-down approaches.

Conclusions:

• GluC-based middle-down MS is a viable method for H4 proteoform quantitation.
• Both top-down and middle-down MS approaches are complementary for comprehensive proteoform analysis.
• This work advances the understanding of histone modifications in genome regulation.