Advancing native top-down MS analysis of non-covalent protein complexes: The Thermo Scientific Q Exactive UHMR mass spectrometer

Importance of the Topic

Native mass spectrometry preserves non covalent interactions enabling direct study of intact protein assemblies in their natural state. It is essential for understanding quaternary structure function relationships, ligand binding, and the assembly of macromolecular complexes in structural biology, drug discovery, and industrial quality control.

Study Objectives and Overview

This study examines the innovations of the Thermo Scientific Q Exactive UHMR Hybrid Quadrupole Orbitrap mass spectrometer to extend native top down mass spectrometry capabilities. Model systems include the E coli GroEL chaperonin, rabbit 20S proteasome, multidrug transporter LmrP, and membrane transporter AmtB. The goal is to improve sensitivity, mass range, resolution, and enable pseudo MS3 analysis of non covalent assemblies.

Methodology and Used Instrumentation

The Q Exactive UHMR platform integrates hardware and software upgrades over its predecessor to enhance native MS performance. Key advances include:

• In source trapping via pulsed ion trapping in the injection flatapole to improve desolvation and generate controlled subunit dissociation
• Reduced RF frequencies in ion routing multipoles including injection and bent flatapoles, quadrupole, transfer multipole and HCD cell to boost high m/z ion transmission and sensitivity
• High mass quadrupole operating at lower RF frequency for selection of ions up to m/z 25000
• Increased HCD collision energy range up to 300 V and fine control of voltage ramp in the Orbitrap analyzer for efficient capture of very high m/z ions

This configuration supports native MS1 of intact complexes, in source dissociation for MS2 fragmentation, and quadrupole isolation followed by HCD to yield pseudo MS3 spectra for sequence analysis.

Main Results and Discussion

E coli GroEL 14 mer was analyzed by native MS1 yielding a clear charge envelope. In source MS2 produced stripped complexes and monomer subunits. Quadrupole isolation of the monomer followed by HCD MS3 resulted in 112 b and y ions corresponding to 21 percent sequence coverage.

Rabbit 20S proteasome 28 mer showed baseline resolved signals with a deconvoluted mass of 717023 Da within 0.082 percent of expected. MS2 with stepped HCD energies released alpha subunits. Pseudo MS3 targeting the 16 plus charge state of alpha6 delivered high resolution fragmentation for unambiguous subunit identification. Deconvoluted in source MS2 also resolved six of seven alpha subunits with monoisotopic mass accuracy.

AmtB membrane protein trimer was released from detergent micelles by in source trapping and measured at 126790 Da with low ppm accuracy. Subsequent MS2 fragmentation produced 75 matched b and y ions covering 19 percent of residues.

LmrP multidrug transporter was similarly liberated from detergent and analyzed by native top down MS producing 104 b and y ions for 33 percent coverage.

Benefits and Practical Application of the Method

The UHMR system offers orders of magnitude higher sensitivity for low abundance samples, extended mass range to >25 k m/z, true isotopic resolution of large complexes and subunits, and the ability to perform pseudo MS3 for targeted sequence analysis. It enables structural characterization of heterogeneous and membrane protein assemblies with minimal sample consumption.

Future Trends and Potential Applications

Future developments may integrate ion mobility separation, further extend mass range and resolution, and enhance automation and data analysis workflows. Native top down pseudo MS3 will facilitate studies of multi subunit complexes, virus particles, protein nucleic acid assemblies, ligand binding dynamics, and high throughput structural profiling in biopharma and proteomics.

Conclusion

The Thermo Scientific Q Exactive UHMR mass spectrometer advances native mass spectrometry by combining in source trapping, optimized RF routing, high collision energy, and high resolution Orbitrap detection. It overcomes previous limits in mass range and sensitivity and enables detailed top down analysis of large non covalent protein complexes, expanding capabilities for structural proteomics and biophysical characterization.

References

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• Ben Nissan G et al Analytical Chemistry 2017 89 4708–4715
• Van de Waterbeemd M et al Nature Methods 2017 14 283–286
• Fort K L et al Analyst 2017 143 100–105
• Viner R et al Integrative Structural Proteomics IMSC 2018