Increasing Throughput in Collision-Induced Unfolding Studies: Enhancing Sensitivity and Streamlining Set-up

Significance of the topic

Collision-induced unfolding (CIU) is an increasingly important gas-phase technique for probing the conformational stability and domain architecture of proteins, protein complexes and biotherapeutics. CIU fingerprints provide rapid, diagnostic information on ligand-binding effects, proteoform- or mutation-driven changes, and domain stability that are of high value in biopharmaceutical developability assessment and quality control. Growing interest from industry creates demand for faster, more sensitive and easier-to-deploy CIU workflows that can be integrated into routine analytical pipelines.

Objectives and study overview

This work presents two complementary innovations implemented on the Cyclic IMS P20 mass spectrometer aimed at increasing CIU throughput and sensitivity: (1) dedicated CIU acquisition types (CIU HDMS and CIU HDMSMS) that greatly accelerate method creation and support both multiplexed and single-charge-state experiments; and (2) Wideband Enhancement (WBE), a TOF duty-cycle optimization that substantially increases signal and enables shorter run times. The study demonstrates these capabilities using a streptavidin standard and a Waters humanized monoclonal antibody (mAb) mass-check standard and evaluates impacts on method setup time, signal intensity and experiment duration.

Methods and workflow

The CIU acquisition types allow users to define a starting collision voltage, voltage step size, number of steps and time per step to create a complete CIU experiment rapidly (method creation reported to be >15 times faster than prior manual configuration). Two acquisition sequencing modes are supported: sequential mode (each collision voltage applied for its full duration before advancing) and parallel mode (voltage ramp is cycled scan-by-scan throughout the run), the latter enabling online or LC-coupled CIU experiments. Data were acquired on the Cyclic IMS P20 and processed with MassLynx v4.2 and CIUSuite2 for generation of CIU fingerprints. Typical CIU workflows require many voltage steps to resolve discrete unfolding transitions; WBE was evaluated as a means to recover signal loss associated with fast or multiplexed acquisition.

Used instrumentation

• Cyclic IMS P20 Mass Spectrometer with high-resolution cyclic ion mobility separator and TOF mass analyzer capable of ~100,000 resolving power (FWHM).
• Optional modules available on the platform: Dynamic Field Declustering (DFD) for native MS adduct/detergent removal and electron capture dissociation (ECD) for complementary fragmentation-based studies.
• Ion source and front-end: Waters Nanolockspray ionization source with nanoflow capillary adaptor.
• Software: MassLynx v4.2 for acquisition and CIUSuite2 for CIU data processing and fingerprint visualization.
• Test samples: Streptavidin (Pierce) and Waters Humanized mAb mass check standard.

Main results and discussion

• Method setup time: The CIU HDMS and CIU HDMSMS acquisition templates reduce manual method creation time by at least 15-fold, enabling methods to be configured in under one minute by entering start voltage, step size, number of steps and time per step.
• Sensitivity gains with Wideband Enhancement: WBE increases the TOF sampling duty cycle and produced approximately a fourfold signal increase for the 15+ charge state of streptavidin and greater than fourfold for the 26+ charge state of the mAb standard. This increased ion throughput enables substantial reductions in acquisition time while preserving the quality of CIU fingerprints.
• Throughput improvements: Because CIU experiments conventionally require many voltage steps (leading to total experiment times of 15 minutes or longer), the combination of accelerated method set-up and the sensitivity gain from WBE can reduce total run times by up to ~4x, directly increasing sample throughput.
• Acquisition flexibility: Sequential vs. parallel CIU modes provide operational flexibility. Parallel (scan-by-scan) cycling supports the possibility of LC-coupled online CIU experiments, expanding applicability to higher-throughput chromatographic workflows.
• Data analysis: CIUSuite2 was used to generate and compare CIU fingerprints; increased signal from WBE improved the clarity of arrival time distributions and resolved unfolding transitions for higher charge states.

Benefits and practical applications

• Faster experiment design and deployment reduces operator time and facilitates unsupervised batch CIU analyses.
• Higher sensitivity permits shorter acquisitions or lower sample consumption while maintaining diagnostic unfolding detail—beneficial for limited or precious samples such as therapeutic mAbs and complexes.
• Compatibility with LC workflows through parallel acquisition mode opens the door to online CIU screening during separations, useful for developability studies and routine QA/QC.
• Neutral to positive impact on data quality: improved duty cycle enhances signal-to-noise for high-charge-state ions that are most informative in CIU.
• Modularity of the platform (DFD, ECD options) allows combining native MS, CIU and fragmentation studies for comprehensive biotherapeutic characterization.

Future trends and potential applications

• Integration of rapid CIU templates and duty-cycle improvements into automated, high-throughput pipelines for biopharma developability screening and lot-release testing.
• Wider adoption of online LC-CIU workflows for separations of complex mixtures, enabling structure-stability mapping across chromatographic peaks in a single run.
• Combination of WBE with other sensitivity and ion-utilization strategies (e.g., improved ion optics, trapping, multiplexed IMS) to further compress experiment times without losing structural resolution.
• Advanced data-processing and machine-learning tools applied to high-throughput CIU fingerprints for automated classification of stability profiles, formulation effects and critical quality attributes.
• Application extensions to larger assemblies, membrane proteins and heterogeneous glycoforms where sensitivity gains will be particularly valuable.

Conclusion

The Cyclic IMS P20 platform introduces two practical innovations that together address key throughput and sensitivity bottlenecks in CIU studies: dedicated CIU acquisition templates that drastically accelerate method creation, and Wideband Enhancement that increases TOF duty cycle and delivers roughly fourfold signal gains for representative protein charge states. Combined, these advances enable up to approximately fourfold reductions in CIU run time, broaden the feasibility of online CIU acquisitions, and facilitate higher-throughput structural screening workflows relevant to biopharmaceutical development and routine analysis.

Reference

Elliott N., McCullough B., Marsden-Edwards E., Cooper-Shepherd D. A. Increasing Throughput in Collision-Induced Unfolding Studies: Enhancing Sensitivity and Streamlining Set-up. Waters Corporation poster, 2026.