Extending the mass limit of collision cross sections of proteins in Orbitrap analyzers through kinetic energy and fragmentation behavior analysis

Significance of the topic

Proteins adopt specific gas-phase structures that influence their biological function, making accurate measurement of collision cross sections (CCS) critical for understanding protein conformations and interactions.

Objectives and Study Overview

This work aims to extend the mass range for CCS measurements using Orbitrap mass analyzers by investigating the role of ion kinetic energy and fragmentation behavior, and to benchmark Orbitrap-derived CCS values against conventional ion mobility (IM) data for proteins up to 50 kDa under both denaturing and native-like conditions.

Methodology

• Orbitrap CCS measurement is based on analyzing the decay rate of ion packet oscillation amplitudes due to collisions with neutral gas in the Orbitrap analyzer.
• The decay constant is extracted by transforming time-domain signals using FFT and fitting exponential decay to derive collision frequency, which is related to CCS through calibration.
• Experiments were performed on a modified QE High Field Orbitrap using transients up to 2 seconds to capture sufficient signal beats for accurate decay fitting.
• Protein standards ranging from 19.5 kDa to 37 kDa were analyzed in various charge states under denaturing and native-like conditions.

Used Instrumentation

• Modified QE High Field Orbitrap mass spectrometer with extended transient acquisition capability.
• Electrospray ionization source.
• Quadrupole mass filter, C-trap, collision cell and optimized ion optics for gas-phase ion trapping.
• Nitrogen as collision gas in the Orbitrap analyzer.

Main Results and Discussion

• Orbitrap-derived CCS values agree closely with IM measurements for smaller proteins (<16 kDa) and high-charge-state larger proteins (20–50 kDa) sprayed from denaturing solutions.
• Low-charge-state ions exhibit systematic underestimation of CCS due to reduced kinetic energy leading to ion survival after collisions and hence slower decay rates.
• Analysis of minimum kinetic energy and collision energy thresholds revealed a linear correlation between protein mass and the energy required to remove ions from the packet upon single collision.
• Regression models predict energy thresholds as a function of mass and charge, providing a framework to correct CCS measurements for low-charge-state ions.

Benefits and Practical Applications

• Extended Orbitrap CCS measurements enable direct assessment of larger protein conformations without the need for separate ion mobility devices.
• Method facilitates native protein complex analysis in structural biology, quality control in biopharmaceutical development, and proteomics workflows.
• High-resolution Orbitrap CCS data enhance the capability for conformational profiling and ligand-binding studies in the gas phase.

Future Trends and Potential Applications

The integration of kinetic energy threshold models may allow automated correction of Orbitrap CCS data for a broader range of proteins and complexes. Future investigations will explore structural stability, binding energetics of protein assemblies, and expansion to megadalton-scale analytes using single-ion tracking techniques.

Conclusion

Accurate collision cross-section measurements in an Orbitrap analyzer depend on sufficient transient length and consideration of ion kinetic energy. While high-charge-state protein CCS values align with ion mobility benchmarks, low-charge-state ions require energy-based corrections. The presented kinetic energy analysis framework advances the applicability of Orbitrap CCS methods to larger, native-like protein assemblies.

References

• Sanders JD, Grinfeld D, Aizikov K, Makarov A, Holden DD, Brodbelt JS. Determination of Collision Cross-Sections of Protein Ions in an Orbitrap Mass Analyzer. Anal Chem. 2018;90(9):5896–5902.
• Makarov A, Denisov E. Dynamics of Ions of Intact Proteins in the Orbitrap Mass Analyzer. J Am Soc Mass Spectrom. 2009;20(8):1486–1495.
• Bush MF, Hall Z, Giles K, Hoyes J, Robinson CV, Ruotolo BT. Collision Cross Sections of Proteins and Complexes Measured by Ion Mobility-Mass Spectrometry and Orbitrap-Based Methods. Anal Chem. 2010;82(22):9557–9565.
• Wörner TP, Aizikov K, Snijder J, Fort KL, Makarov AA, Heck AJR. Frequency Chasing of Individual Megadalton Ions in an Orbitrap Analyzer Improves Precision of Analysis in Single Molecule Mass Spectrometry. bioRxiv. 2021;2021.06.15.448530.
• Dziekonski ET, Johnson JT, Lee KW, McLuckey SA. J Am Soc Mass Spectrom. 2018;29(2):242–250.
• Jiang T, Chen Y, Mao L, Marshall AG, Xu W. Phys Chem Chem Phys. 2015;18(2):713–717.
• Yang F, Voelkel JE, Dearden DV. Anal Chem. 2012;84(11):4851–4857.