Advances in Orbitrap Instrumentation for Native Top-Down Analysis of Non-Covalent Protein Complexes

Significance of the topic

Native mass spectrometry combined with top-down analysis offers direct insight into non-covalent protein assemblies in their near-native state. This approach is crucial for understanding multimeric structures, protein–protein and membrane protein interactions, and for advancing structural proteomics and drug discovery applications.

Objectives and study overview

The study evaluates performance enhancements of the new Thermo Scientific Q Exactive UHMR for native MS and pseudo-MS3 top-down analysis of large protein complexes. Modifications aim to overcome challenges in desolvation, high m/z transmission, and fragmentation efficiency, extending analysis to heteromeric assemblies and membrane proteins.

Methodology

A series of native MS and pseudo-MS3 experiments were performed on model systems including the GroEL chaperone 14-mer, rabbit 20S proteasome, the LmrP multidrug transporter, and the AmtB membrane protein trimer. Key hardware and software adjustments were implemented to improve ion desolvation and transmission.

Instrumentation Used

• Q Exactive UHMR hybrid quadrupole-Orbitrap mass spectrometer
• In-source trapping in the injection flatapole
• Reduced RF frequencies in injection/bent flatapoles, quadrupole, C-trap, and HCD cell
• Enhanced HCD collision energy (up to 300 V)
• Optimized gas pressure and Orbitrap voltage ramp rate

Main results and discussion

Enhancements yielded:

• Efficient removal of detergent micelles and improved desolvation allowing accurate intact mass determination (e.g., AmtB trimer)
• Robust transmission of high m/z ions enabling clear MS1 envelopes for megadalton assemblies
• Enhanced fragmentation into monomers, stripped complexes, and sequence ions: identification of up to 104 b/y ions (25 % sequence coverage) for 20S proteasome and 75 matched ions (19 % coverage) for AmtB
• Mass accuracy within 0.082 % deviation from theoretical mass

Benefits and practical applications

The upgraded platform facilitates structural characterization of both soluble and membrane-bound protein complexes, streamlines analysis workflow for biopharmaceutical quality control, and supports structural biology studies of complex assemblies under native conditions.

Future trends and opportunities

Ongoing developments may include integration with ion mobility separation, higher-energy dissociation strategies for improved sequence coverage, automation for high-throughput structural proteomics, and applications to ever-larger and more heterogeneous complexes.

Conclusion

The modified Q Exactive UHMR demonstrates outstanding capability for native top-down analysis of non-covalent protein assemblies, overcoming key technical hurdles in desolvation, transmission, and fragmentation, and opening new avenues for detailed structural studies of complex biomolecular systems.

References

1. Belov ME et al. Anal Chem. 2013 Dec 3;85(23):11163-73.
2. Ben-Nissan G et al. Anal Chem. 2017 Apr 18;89(8):4708-15.
3. van de Waterbeemd M et al. Nat Methods. 2017 Mar;14(3):283-286.
4. Fort KL et al. Analyst. 2017 Dec 18;143(1):100-105.