Studying protein-drug complexes under native conditions in the low nM range using MRMS

Significance of the Topic

This work highlights the critical role of native mass spectrometry in preserving weak, non-covalent protein–ligand interactions under conditions that closely mimic the physiological environment. Achieving detection and quantitation of binding events in the low nanomolar range is vital for early-stage drug discovery, enabling accurate assessment of affinity and specificity without requiring chemical modifications or labels.

Study Objectives and Overview

The primary aim was to demonstrate how a magnetic resonance mass spectrometer (scimaX® MRMS) can be applied to measure the association between carbonic anhydrase II (CA II) and the inhibitor acetazolamide. Key goals included:

• Recording isotopically resolved mass spectra of the free enzyme and its drug complex under native, gentle desolvation conditions.
• Calculating the equilibrium dissociation constant (K_D) in the low nanomolar range.
• Verifying mass accuracy for both protein and complex through high-resolution deconvolution methods.

Methodology and Instrumentation

Samples of bovine CA II and the small-molecule ligand acetazolamide were prepared in ammonium acetate buffer at pH 7.0. Protein concentrations ranged from 5 to 20 nM, while ligand levels spanned 2–800 nM. After mixing and brief incubation, solutions were infused via electrospray ionization.

Used Instrumentation

The scimaX® MRMS platform (Bruker Daltonik GmbH) operated in positive-ion mode with externally calibrated spectra. Conditions included a flow rate of 4 µL/min, capillary voltage of –4.5 kV, drying gas at 200 °C, and a resolving power of ~70 000 at m/z 2645. Data acquisition summed 200 scans with a 7 s ion accumulation time.

Data Processing

Deconvolution of charge states 10+, 11+ and 12+ was carried out using MaxEnt (DataAnalysis 5.2). Monoisotopic mass assignments employed SNAP2, and equilibrium binding curves were fitted with non-linear regression in PRISM software to extract K_D values.

Main Results and Discussion

Fully isotopically resolved spectra confirmed distinct peaks for free CA II (≈29 070 Da) and the CA II–acetazolamide complex (≈29 292 Da). Mass differences yielded ligand mass within a few millidaltons of the known 221.9881 Da. Binding isotherms at protein levels of 5 nM and 20 nM gave K_D values of 43 ± 7 nM and 52 ± 12 nM, respectively, closely matching literature reports (~20 nM for human CA II).

Benefits and Practical Applications

Native MRMS delivers several advantages for biochemical and pharmaceutical research:

• Direct, label-free visualization of all species in solution, including isoforms and non-specific adducts.
• Quantitative determination of binding affinities down to low nanomolar dissociation constants.
• Capability to operate in high-salt buffers without compromising native conformations.
• Potential to streamline primary and secondary screening workflows in drug development.

Future Trends and Potential Applications

Advances in native MRMS are expected to broaden its impact across several domains:

• Integration into high-throughput fragment screening and lead optimization pipelines.
• Expanded analysis of larger protein complexes and multi-component assemblies.
• Automation of sample handling for routine, label-free binding assays.
• Coupling with computational docking and molecular modeling for detailed structure–function correlations.

Conclusion

The scimaX® MRMS approach offers unmatched sensitivity and resolution for native analysis of protein–ligand complexes at physiological ionic strength and low nanomolar concentrations. Its capacity to deliver accurate masses, isotopic distributions, and binding constants in a single experiment makes it a powerful tool for medicinal chemistry and structural biology.

References

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