TANDEM ION MOBILITY COUPLED WITH MASS SPECTROMETRY FOR GAS PHASE UNFOLDING STUDIES

Significance of the Topic

The ability to characterize protein structures and unfolding pathways in the gas phase using tandem ion mobility spectrometry coupled with mass spectrometry (IMS-MS) has become a cornerstone in structural proteomics. It enables separation of conformational ensembles, measurement of collision cross sections, and detailed probing of domain stability via collision-induced unfolding (CIU).

Objectives and Study Overview

This work presents a custom cyclic IMS-enabled Q-Tof platform with multiple IMS stages (IMSn) and intermediate activation steps. The main goal is to illustrate its flexibility in performing sequential mobility separations and controlled unfolding of protein subpopulations in the gas phase. Human transthyretin (TTR) tetramer serves as a model system to validate stepwise CIU and advanced IMSn experiments.

Applied Instrumentation

• ESI-Q-cIM-Tof system with extended time-of-flight analyzer and dual gain ADC
• Cyclic ion mobility (cIM) device providing a single-pass path length of 98 cm and adjustable T-wave arrays for resolution beyond 200 (Ω/ΔΩ)
• Segmented quadrupole transfer ion guide and stacked-ring ion guides for ion trapping and activation
• Nitrogen drift gas and helium within the cIM entrance cell; trap collision cell for CIU experiments

Methodology

Human TTR was buffer exchanged into 200 mM ammonium acetate and infused via nanoelectrospray ionization. Native mass spectra were recorded followed by single-pass cIM separation. Collision-induced unfolding was initiated in the trap cell at elevated voltages (e.g., 40 V), generating multiple unfolded conformers. IMS2 and IMS3 workflows incorporated mobility selection, ejection to a pre-store, reinjection, and additional activation steps to track unfolding pathways of isolated populations.

Key Results and Discussion

Single-pass cIM of the native 16+ TTR tetramer yielded a single conformational family per charge state. CIU in the trap cell produced at least five distinct unfolded states. IMS2 experiments allowed isolation of specific conformers (labeled species 1–4), each undergoing further unfolding upon reinjection and activation. IMS3 demonstrated that multi-stage analyses can map sequential transitions, revealing only lower-mobility species and no refolding collapse under the studied conditions. The platform’s software enabled flexible selection of arrival time windows and multiple mobility selections per cycle.

Benefits and Practical Applications

The cyclic IMS-enabled Q-Tof offers high resolution, rapid cycling for multi-pass experiments, and user-defined mobility selection. Its ability to combine IMSn with in-line activation provides a detailed view of protein unfolding landscapes. This approach benefits structural biology, quality control of biotherapeutics, ligand-binding studies, and fundamental investigations of gas-phase protein dynamics.

Future Trends and Applications

Anticipated developments include integration with alternative activation methods (e.g., UVPD, ECD), automated multi-stage IMS workflows, and coupling with machine learning for conformational assignment. Expansion to larger complexes, membrane proteins, and ligand screening assays will further enhance the utility of IMSn for comprehensive structural analysis.

Conclusion

The custom cyclic IMS-enabled Q-Tof system successfully demonstrated multi-stage collision-induced unfolding of the TTR tetramer, reproducing known unfolding pathways (IMS2) and extending analysis through IMS3. Its flexible mobility selection and activation scheme provide a powerful platform for detailed gas-phase structural studies.

References

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