BOOSTING SENSITIVITY IN TOP-DOWN PROTEIN STUDIES USING CYCLIC IMS-ENABLED WIDEBAND ENHANCEMENT

Significance of the topic

The ability to characterize intact proteins and their post-translational modifications (PTMs) by top-down mass spectrometry depends critically on MS/MS sensitivity. For intact proteins, signal is dispersed across many product ions, multiple charge states and broad isotope envelopes, reducing per-ion intensity and limiting sequence coverage and confident PTM localization. Methods that increase MS/MS duty cycle and ion utilization therefore directly improve the depth and reliability of top-down analyses, particularly for low-efficiency fragmentation methods such as electron capture dissociation (ECD).

Objectives and overview of the study

This work reports the implementation and evaluation of Wideband Enhancement (WBE) on a Cyclic IMS P20 time-of-flight mass spectrometer to boost MS/MS sensitivity in top-down protein experiments. The study aimed to quantify sensitivity gains for both collision-induced dissociation (CID) and ECD across the mass range, demonstrate compatibility with ion mobility separation, and assess the impact on sequence coverage for small and medium-sized proteins (bovine ubiquitin and bovine carbonic anhydrase II). Experiments compared single-pass IMS, bypass IMS, and TOF-only acquisition with and without WBE.

Methods and used instrumentation

• Samples: Bovine ubiquitin (~8.5 kDa) and bovine carbonic anhydrase II (~29 kDa) analyzed under denaturing conditions by direct infusion.
• Instrument: Waters Cyclic IMS P20 mass spectrometer equipped with an electron capture dissociation cell; positive electrospray ionization (ESI) operation.
• Fragmentation: Both CID and ECD MS/MS were used to generate complementary fragment ion series (b/y and c/z types).
• Ion mobility modes: Single-pass mode (0.98 m cyclic travelling-wave separation) and bypass mode (minimal separation) were tested; multi-pass mobility acquisitions were also considered for enhanced separation.
• Wideband Enhancement (WBE): Synchronization of ion packets exiting the IMS with the TOF pusher to maximize duty cycle for defined m/z windows, increasing the fraction of ions detected.
• Data acquisition and processing: MassLynx v4.2 for acquisition; DriftScope, waters_connect and UNIFI peptide mapping workflows for visualization and sequence coverage analysis.

Main results and discussion

• Sensitivity gains: WBE produced up to a 10-fold increase in fragment ion intensity for top-down MS/MS. Representative improvements include ~10× for low m/z fragments and ~8× for high m/z fragments in ubiquitin CID spectra.
• ECD performance: Because ECD has lower intrinsic fragmentation efficiency than CID, WBE provided particularly pronounced benefits. For carbonic anhydrase II, ECD sequence coverage improved from 54% (WBE off) to 70% (WBE on) in bypass IMS mode, a ~15 percentage point gain, enabling denser c- and z-type ion series across the mass range.
• IMS compatibility: WBE enables high-sensitivity MS/MS while retaining ion mobility separation. Bypass IMS combined with WBE yielded the highest sensitivity boost, but single-pass IMS with WBE also provided significant gains without sacrificing spectral resolution.
• Practical spectral effects: WBE not only increased peak intensities but improved peak profiles and signal-to-noise, facilitating detection of low-abundance fragments and better coverage of isotopic clusters.

Benefits and practical applications

• Enhanced top-down proteomics sensitivity: WBE increases the detectable fragment ion population, improving identification confidence and enabling detection of low-abundance proteoforms and PTMs.
• Improved ECD utility: By amplifying low-efficiency ECD fragments, WBE makes electron-based dissociation more practical for larger proteins and more informative for backbone cleavage mapping.
• Versatility with IMS: The approach preserves or complements ion mobility separation, so structural/shape information can be retained alongside improved MS/MS sensitivity.
• Laboratory impact: Greater sensitivity allows reduced sample amounts, faster acquisition or higher throughput in targeted top-down workflows, beneficial for research and QA/QC applications.

Future trends and possibilities for use

• LC–MS integration: Translating WBE gains from direct infusion into LC-coupled top-down workflows to improve chromatographic peak interrogation and proteoform profiling in complex samples.
• Adaptive acquisition: Real-time control of WBE windows guided by mobility or precursor information to optimize duty cycle for dynamically changing m/z distributions.
• Extension to other electron-based methods: Applying WBE concepts to ETD and hybrid electron-driven techniques to similarly boost sensitivity for complementary fragmentation channels.
• Native and larger-protein analysis: Leveraging improved ion utilization to expand intact-protein coverage under native conditions and for higher-mass proteoforms.
• Automation and AI: Combining WBE-enhanced data with automated interpretation and machine-learning tools to accelerate proteoform discovery and PTM localization.

Conclusion

Wideband Enhancement implemented on a Cyclic IMS P20 mass spectrometer markedly increases MS/MS sensitivity for top-down protein analysis, delivering up to 10× higher fragment ion signals while remaining compatible with ion mobility separation. The approach is especially valuable for electron-based fragmentation (ECD), where it substantially improves sequence coverage for larger proteins. WBE therefore provides a practical route to more comprehensive and confident intact-protein characterization in research and applied laboratory settings.

References

1. Cui W, Rohrs HW, Gross ML. Top-down mass spectrometry: Recent developments, applications and perspectives. Analyst. 2011;136(19):3854–3864.
2. Hoang C, Uritboonthai W, Hoang L, et al. Tandem Mass Spectrometry across Platforms. Analytical Chemistry. 2024;96(14):5478–5488.