Top‐Down Mapping of Protein Modifications by Multimodal MSⁿ on the New timsOmni Platform

Significance of the Topic

Histone proteins bear dense arrays of post-translational modifications that coordinate chromatin dynamics and gene regulation. Achieving residue-level mapping of multiple co-occurring acetylations on intact proteoforms is critical for epigenetic research, yet remains analytically challenging due to isomeric species and complex fragmentation requirements.

Study Objectives and Overview

• Implement a multimodal MSn workflow (eXd MS2/MS3) on the new timsOmni platform for top-down proteomics.
• Localize and map multiple lysine acetylation sites on histones H3.1 and H4 following in vitro acetyltransferase reactions.
• Compare fragmentation modes (ECD, RCID, EID) for sequence coverage and PTM site localization.
• Demonstrate consistent PTM mapping across different charge states and enzyme treatments (p300, GCN5, PCAF).

Methodology and Instrumentation

Histone H4 and H3.1 proteoforms were incubated with acetyltransferases (p300, GCN5, PCAF) under buffered conditions. Direct infusion nano-ESI introduced intact proteins into a timsOmni platform integrating trapped ion mobility spectrometry (TIMS) with a segmented Omnitrap linear ion trap. Multimodal fragmentation cascades combined electron capture dissociation (ECD), resonance excitation collision-induced dissociation (RCID) and electron induced dissociation (EID) in MS2 and MS3 to maximize sequence information.

Used Instrumentation

• timsOmni platform (TIMS-Q-TOF with Omnitrap segmented linear ion trap)
• Nano-ESI source for direct infusion
• Electron capture dissociation (ECD), resonance excitation CID (RCID), electron induced dissociation (EID) workflows
• Data acquisition at multiple charge states (z=13+ to 19+)

Key Findings and Discussion

• Combined eXd MS2/MS3 achieved near-100% sequence coverage of histone H4 and H3.1 proteoforms, enabling confident localization of up to seven acetylation sites (K5, K8, K12, K16, K20, K31, K44) on H4(Kac)₆.
• A preferential acetylation pattern emerged: lower acetylation states (1–4 sites) targeted N-terminal tail lysines (K5–K16), while higher states extended into core regions.
• Monoacetylated H3.1 showed exclusive modification at K14, consistent for both GCN5 and PCAF enzyme treatments, confirming enzyme specificity.
• Fragmentation mode complementarity improved PTM site discrimination and resolution of positional isomers across charge states.

Benefits and Practical Applications

The eXd multimodal workflow on timsOmni provides a robust strategy for comprehensive top-down proteoform characterization, offering residue-level PTM mapping essential for epigenetics, biomarker discovery, and quality control in proteomic studies.

Future Trends and Opportunities

• Integration of additional fragmentation chemistries and higher throughput acquisition.
• Automated data analysis pipelines for PTM-laden proteoform libraries.
• Application to clinical epigenetic profiling and dynamic cellular signaling studies.
• Expansion to other PTM types (methylation, phosphorylation) in intact proteins.

Conclusion

Multimodal MSn (eXd) on the timsOmni platform enables nearly complete sequence coverage and precise localization of multiple lysine acetylations on intact histone proteoforms, addressing key analytical challenges in top-down proteomics and epigenetics research.

References

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