DEVELOPMENT OF A MODIFIED CYCLIC IMS PLATFORM FOR ENHANCED BIOMOLECULE CHARACTERIZATION

Importance of the topic

Cyclic ion mobility–mass spectrometry (cIMS–MS) is a leading platform for high-resolution separation of gas-phase biomolecules combined with orthogonal fragmentation techniques. Enhancements that increase ion transmission, selectivity, and sensitivity without compromising mobility resolution directly impact native MS, top-down and bottom-up proteomics, characterization of noncovalent assemblies, and structural workflows such as collision-induced unfolding (CIU) and electron-based dissociation (ECD). The modifications described aim to expand practical capability for large biomolecule characterization by improving declustering, boosting sensitivity across broad m/z ranges, and simplifying CIU method generation.

Objectives and study overview

This work presents hardware and software modifications to the Cyclic IMS P20 mass spectrometer intended to:

• Increase sensitivity selectively across mass and mobility space via a Wideband Enhancement (WBE) acquisition mode.
• Improve declustering and desolvation of native and partially adducted ions using Dynamic Field Declustering (DFD) implemented in a modified StepWave ion guide.
• Streamline CIU experiments through a simplified method builder and automated function table generation.
• Demonstrate the practical impact of these changes with example datasets (streptavidin, lysozyme, ubiquitin) and outline combinations with pre-IMS fragmentation techniques (ECD, CID, SID).

Methodology and instrumentation

Key methodological concepts and instrument configuration:

• Wideband Enhancement (WBE): software-controlled synchronization of the TOF pusher with ion arrival and exit from the IMS. A user-defined lookup table maps start/end m/z ranges to start/end drift times enabling selective duty-cycle increase for targeted regions of the mass–drift space. WBE can be applied in multiple acquisition functions within a single run and combined with pre-IMS fragmentation.
• Dynamic Field Declustering (DFD): a hardware modification to the StepWave ion guide. The second stage contains two parallel plate electrodes above and below the ion beam and ring electrodes to the sides. A tunable square-wave RF potential is applied between the plates, forcing ions into oscillatory motion, increasing path length, collisions, and effective desolvation; functionally acting as a low-pass mobility filter that preferentially transmits compact/stable ions and removes unstable/adducted species.
• CIU workflow improvements: a method builder that takes user inputs (start collision voltage, step size, dwell time, number of steps) and automatically creates acquisition functions for rapid CIU fingerprint generation.
• Platform capabilities: Cyclic IMS P20 with optional pre-IMS Waters ECD cell, extended mass range (> m/z 100,000), and an optional high-mass quadrupole resolving to m/z 32,000. The system supports combinations of CID, ECD, SID and mobility separations.

Main results and discussion

Highlights from the reported data and implied performance gains:

• WBE delivered up to ~10-fold signal enhancement for targeted mass/drift regions across examples. For [glu]-fibrinopeptide B and for fragments produced by CID or ECD, signal increases of up to 10x were observed without loss of mobility resolution.
• WBE is tunable: different WBE settings can selectively enhance specific fragment charge states (e.g., +1 vs +2/+3 fragments from lysozyme +11) within a single acquisition using multiple acquisition functions.
• DFD substantially reduces non-specific adducts and matrix cluster signals. Native streptavidin sprayed from 200 mM ammonium acetate showed progressive spectral cleanup with increasing declustering potential; adduct clusters were removed and peak shapes/resolution improved. At the highest DFD potentials, tetramer dissociation was observed, suggesting use for pseudo-MS3 workflows when combined with downstream fragmentation.
• Combining WBE with ECD improved intensities for c- and z-type fragments across both low and higher m/z ranges and improved sequence coverage for ubiquitin, illustrating particular benefit for fragmentation methods with lower intrinsic fragmentation efficiency (e.g., ECD).
• The automated CIU method builder reduced manual setup time and produced CIU fingerprint data efficiently (example: NIST mAb +26 charge state CIU fingerprint generated via CIUSuite2).

Benefits and practical applications

Practical advantages and use cases illustrated by the work:

• Improved sensitivity: WBE enables targeted sensitivity increases up to an order of magnitude, supporting higher-quality MS/MS for low-abundance fragments and expanding utility of electron-driven fragmentation in sequence confirmation.
• Cleaner native spectra: DFD reduces adduction and matrix clustering, making it easier to resolve proteoforms, subunit composition, and post-translational modification patterns in native MS experiments.
• Flexible acquisition: WBE’s LUT-based mapping of m/z to drift time allows per-region optimization; multiple functions can run in one acquisition to favor different fragment populations.
• Enhanced structural workflows: CIU automation plus DFD/WBE improves throughput and interpretability of conformational/CIU studies and can be combined with CID, ECD, SID to produce richer structural information on protein complexes and large biomolecules.
• Pseudo-MS3 capability: DFD-induced dissociation of labile assemblies can be exploited as an upstream activation step prior to downstream fragmentation, enabling staged fragmentation strategies for complex assemblies.

Instrumentation used

• Cyclic IMS P20 Mass Spectrometer (Waters Corporation) with:
  • Modified StepWave Enhanced Declustering ion guide (declustering plates + ring electrodes, square-wave RF).
  • Optional Waters ECD cell in pre-IMS position.
  • High-mass quadrupole option (resolving to m/z 32,000) and instrument upper mass range rated > m/z 100,000.

Future trends and potential applications

Anticipated directions and opportunities building on these modifications:

• Adaptive acquisition: real-time or feedback-driven WBE parametrization (machine-learning optimization of lookup tables) to maximize signal for unknown or complex samples.
• Integration with advanced fragmentation: coordinated WBE/DFD control to optimize staged activation (pseudo-MS3) and to increase sequence coverage in native top-down workflows.
• Broader structural proteomics: improved sensitivity and declustering will aid analysis of higher-order assemblies, heterogeneous glycoforms, and low-abundance proteoforms in biopharma QA/QC and discovery contexts.
• Automation and high-throughput: combining CIU automation with WBE functions for routine structural screening and comparative fingerprinting across sample sets.
• Method standardization: development of community-accepted parameter sets for WBE and DFD for common classes of biomolecules to improve reproducibility between labs.

Conclusions

The combined software and hardware advances—Wideband Enhancement and Dynamic Field Declustering—extend the capabilities of the Cyclic IMS P20 platform for biomolecule characterization. WBE provides a practical mechanism to increase duty cycle and selectively boost sensitivity across the m/z–drift plane, while DFD materially improves desolvation and spectral clarity for native species. Together with automated CIU workflows and an extended mass range, these developments enable higher-sensitivity tandem MS experiments, improved structural readouts for large biomolecules, and potential pseudo-MS3 workflows. Their modular nature supports integration with existing fragmentation strategies, making the platform more versatile for proteomics, native MS, and complex assembly characterization.

Reference

• McCullough BJ, Cooper-Shepherd DA, Hewitt D, Harker D, Marsden-Edwards E. DEVELOPMENT OF A MODIFIED CYCLIC IMS PLATFORM FOR ENHANCED BIOMOLECULE CHARACTERIZATION. Waters Corporation poster (2026).