Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer

Importance of the Topic

The structural characterization of large and heterogeneous biomolecular assemblies under native conditions is essential for advancing our understanding of protein function, interaction networks, and drug targets. Native mass spectrometry (MS) preserves noncovalent interactions and protein conformation, offering complementary information to crystallography and cryo-EM. Recent technological advances have addressed the sensitivity, mass range, and resolution limitations that have historically constrained native MS, opening new possibilities for analyzing intact proteins, complexes, and membrane assemblies.

Objectives and Study Overview

This whitepaper introduces the Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer, designed to overcome native MS challenges. Key objectives include achieving ultra-high mass range resolution, enhancing sensitivity at high m/z, enabling native top-down sequencing (pseudo-MS3), and facilitating analysis of membrane protein complexes directly from detergent micelles.

Methodology and Instrumentation

Innovations in the Q Exactive UHMR platform include:

• In-source trapping in the injection flatapole to gently desolvate large ions and remove detergents.
• Advanced Quadrupole Technology (AQT) with ultra-high mass selection up to 25,000 m/z and beyond.
• Reduced RF frequencies across ion guides (flatapoles, quadrupole, C-trap, HCD cell) to improve transmission of high m/z ions.
• Efficient energy control for HCD fragmentation, supporting sequential subunit ejection and pseudo-MS3 workflows.

Key Results and Discussion

The Q Exactive UHMR demonstrated:

• Resolution of intact E. coli 70S ribosome (2.316 MDa) at ~36,000 m/z, with mass accuracy within 0.6% of theoretical value and mapping of substoichiometric proteins (e.g., RS1 removal and SRA binding).
• Simultaneous detection of 3 MDa and 4 MDa hepatitis B virus capsids under charge reduction, spanning 27,000–36,000 m/z with well-resolved charge states.
• Native MS/MS of a 4 MDa capsid, using quadrupole isolation and HCD to eject up to 17 subunits and track their mass losses up to 6.5% of the particle mass.
• Native top-down analysis of GroEL tetradecamer: in-source dissociation into monomer, 13-mer, and 12-mer, followed by HCD fragmentation and pseudo-MS3 sequencing yielding 21% residue cleavages.
• Release and characterization of the AmtB membrane protein complex from micelles, accurate mass determination, and top-down fragmentation identifying 19% residue cleavages with low-ppm mass accuracy.

Benefits and Practical Applications

These capabilities enable researchers to:

• Analyze scarce or precious samples at high sensitivity, reducing sample requirements.
• Characterize intact membrane proteins and complexes without the need for extensive detergent removal protocols.
• Perform hypothesis-free structural analysis of heterogeneous assemblies and visualize dynamic binding events.
• Integrate native MS data with complementary biophysical methods for comprehensive structural biology studies.

Future Trends and Opportunities

Continued improvements may include extension of mass range beyond 80,000 m/z, deeper integration with ion mobility separations, and automated native top-down workflows. Combining native MS with advanced data analysis platforms (BioPharma Finder, ProSightPC) and cloud-based instrument monitoring will streamline structural proteomics and drug discovery pipelines. Emerging applications include real-time monitoring of protein assemblies in cell extracts, large-scale mapping of interaction networks, and in situ analysis of membrane-bound targets.

Conclusion

The Thermo Scientific Q Exactive UHMR mass spectrometer represents a significant leap forward for native MS, delivering unparalleled resolution, sensitivity, and top-down sequencing capabilities for large protein assemblies and membrane complexes. By addressing traditional limitations, it empowers researchers to explore biomolecular structure and interactions with new depth and confidence.

Used Instrumentation

• Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer
• In-source trapping flatapole and inter-flatapole lens
• Advanced active beam guide (bent flatapole)
• High-mass quadrupole filter (AQT)
• HCD collision cell
• Orbitrap mass analyzer with extended mass range capabilities