PrSM-Level Side-by-Side Comparison of Online LC-MS Methods with Intact Histone H3 and H4 Proteoforms

Area of Science:

• Proteomics
• Biochemistry
• Mass Spectrometry

Background:

• Histone H3 proteoforms exhibit significant heterogeneity, complicating top-down mass spectrometry analysis.
• Liquid chromatography (LC) coupled with mass spectrometry (MS) is crucial for enhancing detection coverage of proteoforms.

Purpose of the Study:

• To compare the efficacy of different LC-MS techniques for the top-down analysis of histone H3 proteoforms.
• To optimize electron-transfer/high-energy collision dissociation (EThcD) parameters for improved proteoform identification.

Main Methods:

• Comparative analysis of reversed-phase LC-MS (RPLC-MS), dual-gradient WCX/HILIC-MS, and organic-rich WCX/HILIC-MS.
• Top-down mass spectrometry analysis of histone H3.1, H3.2, and H4 proteoforms from HeLa cell extracts.
• Utilized optimized EThcD parameters for enhanced fragmentation and detection.

Main Results:

• Both dual-gradient and organic-rich WCX/HILIC-MS methods resolved intact H3 and H4 proteoforms based on acetylation levels.
• The organic-rich WCX/HILIC-MS method demonstrated superior separation of near-isobaric trimethyl/acetyl H3 proteoforms.
• WCX/HILIC-MS approaches significantly improved the quality, range, reproducibility, and confidence of H3 proteoform identifications compared to RPLC-MS.

Conclusions:

• Optimized WCX/HILIC-MS methods, particularly the organic-rich variant, are highly effective for the top-down analysis of complex histone H3 proteoforms.
• These advanced LC-MS techniques enhance the depth and reliability of histone proteoform characterization.
• The findings provide a more robust platform for studying histone modifications and their biological significance.