Native Mass Spectrometry of Membrane Protein-Ligand Complexes Using the SELECT SERIES™ Cyclic™ IMS

Significance of the Topic

Native mass spectrometry of membrane proteins addresses a critical analytical challenge in pharmaceutical research, as membrane proteins account for approximately 60% of all drug targets. Their hydrophobic nature requires specialized approaches to maintain near-native conditions and to remove detergent micelles for meaningful mass spectral analysis.

Objectives and Study Overview

This study demonstrates the capabilities of the SELECT SERIES™ Cyclic™ IMS Mass Spectrometer for native MS of membrane proteins. Key aims include optimizing declustering conditions, measuring collision cross sections to probe higher-order structure, and detecting small molecule binding in drug-discovery workflows.

Methodology and Instrumentation

Membrane proteins were electrosprayed from ammonium acetate solutions containing detergents above their critical micelle concentration. High-energy collisions in the source region (cone voltage) and trap collision cell were applied to liberate proteins from micelles. Cyclic ion mobility measurements provided collision cross section (CCS) data via the TWIMS calibration tool. Instrumentation details:

• SELECT SERIES™ Cyclic™ IMS Mass Spectrometer
• Source region with adjustable cone voltage
• Trap collision cell for collisional activation
• TWIMS CCS calibration using IMScal software

Main Results and Discussion

1) OmpF membrane protein (4 µM, β-OG detergent):

• Without activation: spectra dominated by detergent clusters and protein–detergent complexes.
• With 150 V cone and 200 V trap voltages: clear charge state distribution (m/z 4400–5600), measured mass 111.2 kDa for [M+23H]23+.
• Optimized voltages minimized unfolding (inset), yielding TWCCSN2 (21+) = 6380 ± 52 Å2 indicative of native-like structure.

2) pfMATE protein (4 µM, C8E4 detergent):

• Optimum settings (160 V cone, 6 V trap) delivered isotopically resolved peaks at 50.5 kDa with resolving power Rp = 70 000 (FWHM).
• Mobility-optimized conditions gave CCS (10+) = 3655 ± 20 Å2 for folded protein.

3) Drug-screening with 133 compound mix:

• Native spectrum revealed a single 372 Da mass shift correlating to a 1:1 ligand binding event.
• Direct mass readout facilitates rapid hit identification and ranking in screening campaigns.

Benefits and Practical Applications

• High-quality native MS spectra of membrane proteins through effective detergent micelle removal.
• Accurate CCS measurements enable monitoring of structural changes and unfolding pathways.
• Direct detection of small molecule binding supports streamlined drug-discovery and fragment screening workflows.

Future Trends and Opportunities

Advances may include high-throughput cyclic IMS screening, integration with cryo-EM and computational modelling for comprehensive structural biology, real-time ligand binding assays, and expansion to complex membrane assemblies and protein interactomes.

Conclusion

The SELECT SERIES™ Cyclic™ IMS Mass Spectrometer offers robust declustering, high-resolution mass analysis, and cyclic ion mobility for detailed structural interrogation of membrane proteins and direct ligand binding detection, enhancing both fundamental studies and pharmaceutical screening applications.

References

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