Top-Down Sequencing of Intact Proteoforms Using the timsOmni Mass Spectrometer: Accurate Determination of Co-Occurring Histone Modifications.

Deep characterization of intact proteoforms remains an analytical challenge in functional proteomics, particularly for heterogeneous multi-site post-translational modifications at distinct amino acid residues. Histones are among the most dynamically and diversely post-translationally modified proteins in eukaryotic cells, carrying multiple, co-occurring, and reversible modifications that can give rise to isomeric proteoform species. Tandem mass spectrometry with multimodal fragmentation capabilities is a promising approach for deep characterization of intact proteoforms, such as modified histones. We applied the novel timsOmni mass spectrometer, which incorporates the Omnitrap platform enabling multimodal MSn workflows using controlled in vitro acetylation of recombinant histones H3.1 and H4 by GCN5, PCAF and p300, followed by analysis of endogenous H4 proteoforms. Complementary MS2 electron- and collision-based dissociation (ECD, EID, RCID and ECciD), together with MS3 strategies, produced complete or near-complete backbone fragmentation of intact protein ions (>92% amino acid sequence coverage). For monoacetylated species generated by the more site-selective lysine acetyltransferases, the dominant proteoform matched the known catalytic preferences of the enzymes (H3.1K14ac for GCN5 and PCAF, and H4K8ac for PCAF), while minor positional isomers were also identified and their relative abundance estimated. In contrast, the broader substrate specificity of p300 produced a wide distribution of H4 proteoforms bearing up to seven acetylated lysine residues. Species carrying six and seven acetylations were characterized by multimodal MS2/MS3 experiments, enabling localization of individual acetylation sites and discrimination of positional isomers. Finally, top-down sequencing of endogenous intact H4 proteoforms from human liver extracts yielded amino acid sequence coverages of 92 to 93% for the most abundant species and confident localization of multiple, distant PTMs (acetylation and methylation). These results demonstrate that multimodal MSn fragmentation of intact proteins supports residue-level assignment of combinatorial histone marks and coexisting positional isomers in controlled and endogenous proteoform mixtures.