Histone Analysis Using Mobility- and Mass-Selected Ultraviolet Photodissociation in Tandem with Ultra-High-Resolution...

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Top-down proteomics using ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (UHR FT-ICR MS) can resolve complex proteoforms.
• Identifying and localizing post-translational modifications (PTMs) on intact proteins remains challenging.

Purpose of the Study:

• To develop an integrated method combining ion mobility, mass selection, and ultraviolet photodissociation (UVPD) for improved top-down proteoform analysis.
• To enhance protein sequence coverage and PTM localization accuracy in complex mixtures.

Main Methods:

• Integration of trapped ion mobility and mass selection prior to UVPD.
• Subsequent analysis of UVPD fragments using UHR FT-ICR MS.
• Application to a bovine histone mixture for proteoform identification and PTM analysis.

Main Results:

• Achieved high protein sequence coverage (up to 77%) and PTM localization with mass accuracy <1 ppm.
• Identified 51 proteoforms from H2B, H2A, and H4 core histones, including various PTMs like acetylation, methylation, and phosphorylation.
• Demonstrated a 2x improvement in duty cycle compared to previous methods.

Conclusions:

• The developed mobility/mass-selected UVPD approach enhances top-down proteoform analysis efficiency and accuracy.
• This method provides a foundation for better spectral decongestion and annotation of UVPD fragments from protein mixtures.
• The technique has broad applicability for proteoform analysis with minimal sample preparation.