Using Charge Detection Mass Spectrometry with an Electrostatic Linear Ion Trap and Heated Inlet for the Analysis of Protein Complexes

Importance of the Topic

The analysis of large, heterogeneous protein complexes is critical for understanding biomolecular assembly, function, and regulation in health and disease. Conventional mass spectrometry techniques often fail to resolve high–mass species and overlapping charge states, limiting insights into noncovalent assemblies such as proteasomes and regulatory particles.

Objectives and Study Overview

This study demonstrates the application of charge detection mass spectrometry (CDMS) combined with an electrostatic linear ion trap (ELIT) and a novel heated inlet to:

• Directly measure individual ion mass and charge for large protein assemblies.
• Enhance structural insights through thermal activation and controlled dissociation.
• Resolve the stoichiometry of proteasome regulatory complexes and the 20S core particle.

Methodology and Used Instrumentation

Samples of recombinant Mycobacterium tuberculosis proteasome core particles (20S CP) and bacterial proteasome activator (Bpa) with and without HspR substrate were buffer exchanged into ammonium acetate. Ions were generated by nano-electrospray ionization in positive mode. Mass analysis employed a prototype Waters Xevo CDMS system featuring:

• An electrostatic linear ion trap for 500 ms trapping time.
• A heated transfer inlet adjustable up to 300 °C for thermal activation.
• Fourier transform signal processing to derive individual ion m/z and charge, enabling direct mass calculation.

Main Results and Discussion

For the 20S core particle:

• Native mass of ~729 kDa closely matched the theoretical 720 kDa.
• At 300 °C inlet heating, asymmetric charge partitioning led to dissociation of one or two α-subunits, evidenced by higher m/z envelopes and distinct mass losses.
• Desolvation improvements reduced adducts and enhanced mass accuracy.

For the Bpa–HspR complex:

• Dodecameric Bpa bound on average 1.7 HspR molecules, confirming predominant 2:1 stoichiometry with minimal heterogeneity.
• Heated inlet induced HspR ejection and charge reduction of apo-Bpa, demonstrating controlled dissociation pathways.

Benefits and Practical Applications

This approach offers:

• Precise mass characterization of high-mass noncovalent complexes.
• Enhanced structural interrogation via thermal activation.
• Improved decision-making for downstream structural studies, such as cryo-EM.

Future Trends and Potential Uses

Integration of thermal activation with CDMS is expected to:

• Enable routine analysis of viral capsids, antibodies, and other large assemblies.
• Facilitate studies on complex stability, folding, and subunit interactions under variable conditions.
• Support high-throughput workflows in structural biology and biopharmaceutical development.

Conclusion

The combination of ELIT-based CDMS with a heated inlet provides a robust platform for direct measurement and controlled dissociation of large protein complexes. This methodology overcomes limitations of conventional MS, delivering accurate stoichiometry and structural insights critical for both fundamental research and applied biopharmaceutical analysis.

References

1. Hogan, J. A.; Jarrold, M. F. J. Am. Soc. Mass Spectrom. 2018, doi:10.1021/jasms.3c00177
2. Bolten, M. et al. Structure 2016, doi:10.1021/j.str.2016.10.008
3. Hu, L. et al. J. Biol. Chem. 2018, doi:10.1074/jbc.RA117.001471