Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Histones are crucial for DNA packaging and are amenable to top-down mass spectrometry.
• Previous work established methods for high-quality MS/MS spectra of intact proteins.

Purpose of the Study:

• To extend existing mass spectrometry techniques for analyzing histone proteoforms.
• To identify unique intact and truncated histone variants and their modifications.

Main Methods:

• Utilized sequential ion/ion reactions in a modified Orbitrap Velos Pro/Elite mass spectrometer.
• Employed data-dependent and targeted HPLC-MS experiments for histone analysis.
• Applied parallel ion parking to resolve overlapping m/z species.

Main Results:

• Identified unique intact histone proteoforms with up to 81% sequence coverage.
• Characterized truncated histone H2A and H2B variants with up to 93% sequence coverage.
• Demonstrated the separation of chromatographically unresolved species using parallel ion parking.

Conclusions:

• Advanced mass spectrometry techniques provide deep insights into histone heterogeneity.
• Identified novel truncated histone H2A proteoforms potentially generated by cathepsin L.
• The methods enable comprehensive characterization of histone modifications and variants.