Activated-Ion Electron Transfer Dissociation Enables Electron-Based Dissociation Following Proton Transfer Charge Reduction

Significance of the Topic

Electron-based dissociation methods such as ETD offer extensive sequence information for intact proteins but lose efficiency at low charge states produced by proton transfer charge reduction (PTCR). Integrating activated‐ion ETD (AI-ETD) with PTCR promises to restore fragmentation performance, enabling high‐resolution top‐down proteomics of complex mixtures.

Objectives and Study Overview

The study aimed to evaluate the applicability of AI-ETD on low‐charge density protein ions generated by PTCR. Three standard proteins (apomyoglobin, carbonic anhydrase, enolase) were purified by PTCR, then subjected to either conventional ETD or AI-ETD. Sequence coverage and fragment ion yields were compared to assess each strategy.

Methodology and Instrumentation

Samples were directly infused into a modified Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer equipped for AI-ETD. Following isolation of the predominant charge state envelope, PTCR was performed in a linear ion trap until ions centered around m/z ~2500. Fragmentation used either ETD alone or concurrent infrared irradiation via a Synrad Firestar CO2 continuous‐wave laser (10.6 µm, up to 8 W). MS^3 spectra were acquired in the Orbitrap at 240,000 resolution.

Main Findings and Discussion

Standalone ETD on PTCR‐treated ions produced almost no sequence‐informative fragments. In contrast, AI-ETD generated abundant c and z• ions across all proteins, restoring sequence coverage to levels comparable with ETD on highly charged precursors (e.g., apomyoglobin coverage increased from <1% to 82%). Minor b and y ions indicated low‐level infrared multiphoton dissociation, though IR power optimization maintained predominant electron‐based fragmentation enhancement.

Benefits and Practical Applications

• Enables electron‐based dissociation on ions with reduced charge, expanding PTCR utility.
• Delivers high sequence coverage for intact proteins in complex mixtures.
• Improves sensitivity and specificity in top‐down proteomics workflows.

Future Trends and Potential Applications

Advances such as ion parking to concentrate ion current, refined IR power control, and integration with high‐throughput separation techniques will further enhance AI-ETD/PTCR. Broader adoption is anticipated in structural proteomics, quality control of biotherapeutics, and comprehensive characterization of proteoforms.

Conclusion

Activated‐ion ETD effectively overcomes the charge‐dependence limitations of conventional ETD following PTCR, delivering substantial sequence information from low‐charge precursors. This approach promises robust top‐down analysis of intact proteins in complex samples.

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