High-Efficiency Electron Capture Dissociation of Peptides and Proteins After Collision-Induced Unfolding and Ion Mobility

Importance of the topic

Collision-induced unfolding (CIU) coupled with ion mobility-mass spectrometry (IM-MS) and electron capture dissociation (ECD) unlocks detailed insights into biomolecular conformation and sequence. By unfolding protein ions in the gas phase and probing them with ECD, researchers can correlate higher-order structural changes with primary sequence information, improving characterization of native and modified proteins. This integrated approach addresses historical limitations of performing electron-based fragmentation after rapid IM separations and extends ECD utility to diverse analytes, from small peptides to intact monoclonal antibodies.

Objectives and study overview

The primary goal was to optimize a prototype “ExD cell” for high-efficiency ECD following CIU and IM separation on an Agilent 6560C IM-QTOF platform. Key aims included:

• Balancing ECD efficiency against ion mobility resolving power through gas pressure and lens voltage tuning.
• Applying CIU-IM-ECD workflows to standard peptides (Substance P), small proteins (ubiquitin, cytochrome c), medium-sized enzymes (carbonic anhydrase) and large immunoglobulins (NISTmAb).
• Investigating how unfolding (via CIU voltages) influences ECD sequence coverage and structure–sequence relationships.

Used Instrumentation

The setup comprised an Agilent 6560C IM-QTOF mass spectrometer modified with a prototype ExD cell positioned after the collision cell. Key components:

• In-source CIU capability controlled by collision cell voltages (20–300 V).
• Drift tube ion mobility separation.
• ECD performed in a fly-through ExD cell with adjustable gas pressure and lens voltages between lens 4 and filament bias.
• Sample introduction via nanospray and AJS sources with syringe pump.
• Data analysis using IM-MS Browser and ExDViewer software.

Methodology

Native and unfolded ions were generated by applying defined CIU voltages and separated by drift time. Selected precursors of different charge states were subjected to ECD in the ExD cell. Sequence coverage maps were generated by matching fragment ions to known protein sequences under restrictive criteria to ensure high confidence assignments. Comparative analyses at gentle versus aggressive ECD tunings evaluated impacts on native structures and disulfide bond cleavage.

Main results and discussion

• Cytochrome c (7+): CIU at 20, 150 and 200 V produced distinct conformers (drift times ~28, 33, 38 ms) with similar ECD coverage; lower intensity in intermediate conformer slightly reduced fragment detection.
• Carbonic anhydrase (11+ and 12+): Multiple unfolding transitions were revealed at increasing CIU voltages, with the most unfolded conformer showing enhanced ECD coverage around residues 180–210, highlighting exposure of the C-terminal zinc-binding region.
• NISTmAb (22+ to 30+): Higher charge states correlated with larger collision cross sections (CCS). Under gentle ECD, only N-terminus and CDR3 fragments were observed due to intact disulfide bonds; aggressive ECD cleaved inter-chain disulfides, improving light-chain sequence coverage.
• Substance P (2+): Two overlapping conformers in IM could not be baseline separated, yet both yielded high-efficiency ECD (5–10 %) with S/N >100 for major fragments, demonstrating the method’s applicability to small peptides and isomer differentiation.

Benefits and practical applications

CIU-IM-ECD provides a direct link between gas-phase conformation and primary sequence, enabling:

• Enhanced sequence coverage for proteins resistant to traditional ECD.
• Characterization of labile modifications and disulfide bonds in native complexes.
• Structural probing of peptides and proteins without ion trapping, suitable for high-throughput analysis.

Future trends and potential applications

Further developments are expected to focus on:

• Automated tuning protocols to maximize ECD efficiency and IM resolution.
• Integration with hydrogen-deuterium exchange and other gas-phase structural probes.
• Top-down proteomics workflows for mapping post-translational modifications in complex mixtures.
• Enhanced separation of isomeric species and real-time structural screening in biopharmaceutical QC.

Conclusion

The modified Agilent 6560C IM-QTOF with a fly-through ExD cell demonstrates efficient ECD without compromising IM resolution. Combining CIU, IM separation and ECD enables detailed mapping of protein unfolding pathways and sequence information across a range of analytes from small peptides to intact antibodies. This platform represents a versatile tool for advanced structural proteomics and biotherapeutic characterization.

References

1. Zhang Y, et al. Native electrospray ionization and electron-capture dissociation for comparison of protein structure in solution and the gas phase. Int J Mass Spectrom. 2013;354-355.