EXAMINING THE M/Z SEPARATIVE CAPABILITY OF TRAVELLING WAVES FOR LARGE MOLECULE CHARACTERISATION

Significance of the topic

Traveling wave ion mobility separation extends traditional IM–MS by introducing m/z-dependent transport, enabling analysis of high-mass biomolecules that are challenging to resolve by mobility alone.

Objectives and Study Overview

• Assess the ability of traveling waves to separate ions by m/z in Cyclic IMS and SYNAPT XS instruments.
• Compare TW-IMS separations with linear drift tube (DTIM) and ToF-MS measurements.
• Apply the approach to complex assemblies: a monoclonal antibody (NIST mAb) and adeno-associated virus (AAV) capsids.

Methodology and Instrumentation

Samples were nanoESI-infused into:

• SELECT SERIES Cyclic IMS (Q-TWIM-ToF configuration)
• SYNAPT XS with TW mobility cell, later replaced by a linear field drift tube for pure mobility comparisons

TW parameters (velocity, amplitude) were systematically varied at ~1–2 mbar N₂. mAb ions were activated via elevated cone voltage and collision energy. Multi-pass cyclic IMS and SIMION simulations supported mechanistic insights.

Main Results and Discussion

• Velocity relaxation theory rationalized m/z contributions to TW separations via dimensionless parameters α and γ.
• Distinct mAb charge states were resolved at optimized TW velocities; high TW speed caused unexpected arrival time inversions requiring further study.
• Calibration of TW arrival times against ToF-MS yielded linear correlations for m/z determination.
• Multi-pass cyclic IMS achieved resolution comparable to ToF-MS, limited by analyte heterogeneity.
• TWs enabled separation of empty and full AAV capsids by m/z, while DTIM showed only mobility overlap.
• SIMION simulations suggested observed intermediate signals may arise from capsid-like species of lower density.
• Preliminary CCS estimates (~57 600–63 700 Ų) and radii (~135–142 Å) agreed with cryo-EM data.

Benefits and Practical Applications

• Gentle, gas-phase mass measurement of large, heterogeneous complexes without extensive calibration.
• Relative quantification of viral vector populations for gene therapy development.
• Enhanced charge state resolution for protein higher-order structure analysis.

Future Trends and Potential Applications

• Development of robust TW-MS calibration protocols for routine mass assignment.
• Integration of cyclic IMS with advanced MS platforms for high-throughput large-molecule analysis.
• Extension to other viral vectors, protein complexes, and conformer separations.
• Refinement of ion trajectory models to predict density-dependent species.

Conclusion

Traveling wave-enabled m/z separation at mbar pressures offers a complementary route to drift tube IMS and ToF-MS for characterizing large biomolecular assemblies, demonstrating clear advantages in resolving charge states and viral capsid forms.

References

1. Johnsen R.; Biondi M. A. J. Chem. Phys. 1972, 57 (5), 1975–1979.
2. Richardson K.; Langridge D.; Dixit S. M.; Ruotolo B. Anal. Chem. 2021, 93 (7), 3542–3550.
3. Richardson K.; Langridge D.; Giles K. IJMS 2018, 428, 71–80.
4. Pierson E. E.; Keifer D. Z.; Asokan A.; Jarrold M. F. Anal. Chem. 2016, 88 (13).
5. Silveira M. A.; Large E. E.; Zane G. M.; White T. A.; Chapman M. S. Viruses 2020, 12, 1326.