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

Ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (UHR FT-ICR MS) for top-down proteomics has shown the potential to resolve proteoforms and splice variants, particularly those with post-translational modifications. Here, we integrate trapped ion mobility and mass selection in tandem with ultraviolet photodissociation (UVPD) followed by FT-ICR MS measurements. The proposed method using mobility/mass-selected UVPD before FT-ICR MS allows for high protein sequence coverage and PTM localization with high mass accuracy (<1 ppm) and a better duty cycle (2×). When applied to the analysis of a bovine histone mixture, characteristic UVPD <i>a</i>/<i>b</i>/<i>c</i>/<i>x</i>/<i>y</i>/<i>z</i> ions led to the annotation of 51 proteoforms from H2B, H2A, and H4 core histones with high sequence coverages (up to 77%). Histone variants and PTM combinations, including acetylation, mono-, di-, and trimethylation, and phosphorylation, identified at the top-down level were confirmed using bottom-up analysis. This work provides the foundation for effective mobility and mass preselection of precursor ions and better annotation and spectral decongestion of UVPD fragments from protein mixtures, with general applicability for top-down proteoform analysis with minimal sample preparation.