Top-down Characterization of Intact Proteins via On-line Charge-stripping and Electron Capture Dissociation

Significance of the topic

Characterizing intact proteins with high sequence coverage and structural fidelity is central to proteomics, structural biology and biopharmaceutical analysis. Top‐down electron capture dissociation (ECD) delivers detailed fragmentation patterns, but denaturing conditions produce broad charge state envelopes that limit signal intensity per precursor. Introducing a rapid on‐line chemical charge‐stripping approach improves ECD efficiency by concentrating ion signal into a few low charge states while preserving unfolded conformations for detailed structural and sequence analysis.

Objectives and study overview

This work aimed to integrate microdroplet charge stripping into an electrospray ionization source, couple it with ECD on Q‐TOF instruments, and use ion mobility spectrometry (IM) to correlate gas‐phase structures with fragmentation performance. Specific goals included:

• Developing an on‐line charge stripping method employing dimethylaminopropylamine.
• Evaluating ECD efficiency and sequence coverage for charge‐reduced precursors.
• Using IM‐MS to compare native, denatured and charge‐stripped protein conformations.

Methodology

Proteins ranging from ubiquitin to monoclonal antibodies were prepared under native or denaturing conditions. Denatured samples contained acetonitrile and formic acid, while native samples used ammonium acetate buffers. A dilute solution of dimethylaminopropylamine was infused at controlled flow rates into the reference nebulizer of a Dual Agilent Jet Stream source to induce charge‐stripping reactions in microdroplets. Collisional activation experiments (CIU and CID) were performed by adjusting voltages between capillary exit and fragmentor or collision cell.

Instrumentation

Experiments were conducted on Agilent 6560 and 6545XT AdvanceBio LC/Q‐TOF mass spectrometers modified with a second‐generation ExD cell comprising seven electrostatic lenses, two ring magnets and an electron‐emitting filament. ExD Control software managed cell voltages and tuning. Data were processed using IM MS Browser, ExDViewer for sequence coverage and ECD efficiency, and imsRealTime for IM‐ECD optimization.

Main Results and Discussion

On‐line charge stripping shifted denatured ubiquitin from broad envelopes (6+ to 13+) to focused 2+ to 4+ precursors, enhancing ECD signal intensity. For example, 3+ ubiquitin achieved 100 percent sequence coverage with complementary 100 V CID activation. IM‐MS distinguished compact native and intermediate folded species from elongated charge‐stripped ions. CIU experiments revealed that native 6+ ubiquitin unfolds into a gas‐phase intermediate before adopting more extended structures, whereas charge‐stripped ions predominantly populate elongated conformers. ECD fragmentation patterns of native ions matched known crystallographic B‐factor regions, validating structural preservation.

Benefits and Practical Applications

• Enhanced sequence coverage for low‐charge precursors improves identification of intact proteins and post‐translational modifications.
• Retention of structural information enables comparative studies between solution and gas‐phase conformers.
• Rapid on‐line workflow integrates seamlessly with existing LC/Q‐TOF platforms for high‐throughput analysis.

Future Trends and Opportunities

Emerging directions include coupling charge stripping with liquid chromatography for complex mixtures, extending to larger proteins and multi‐subunit assemblies, and exploring alternative chemical reagents for tailored charge manipulation. Integration with complementary fragmentation methods and automation will further expand applications in proteomics, structural biology and biopharmaceutical quality control.

Conclusion

On‐line microdroplet charge stripping combined with ECD and IM‐MS provides a powerful platform for top‐down characterization of intact proteins. By concentrating ion signal into low charge states and preserving gas‐phase structures, the approach achieves comprehensive sequence coverage and structural insights, offering broad utility for advanced protein analysis.

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.