EXPANDING NATIVE MASS SPECTROMETRY CAPABILITIES FOR SOLUBLE AND MEMBRANE PROTEINS USING A QUADRUPOLE-ION MOBILITY-TIME-OF-FLIGHT MASS SPECTROMETRY SYSTEM

Importance of the Topic

Native mass spectrometry combined with ion mobility provides detailed insights into protein conformations, non‐covalent assemblies, and structural dynamics. This approach is especially critical for membrane proteins, which represent 30 % of the human proteome and over 60 % of drug targets but are challenging to analyze due to their hydrophobicity and detergent requirements.

Objectives and Overview of the Article

This study presents a quadrupole-ion mobility-time-of-flight platform designed to expand native MS workflows for both soluble and membrane proteins. Key aims include demonstrating complex-down sequencing, high-resolution mobility separation, efficient detergent micelle removal, precise collision cross section (CCS) calibration, and automated surface-induced dissociation (SID).

Methodology and Instrumentation

All experiments utilized a SELECT SERIES Cyclic IMS mass spectrometer equipped with a pre-mobility SID device and an ExD cell. Proteins were electrosprayed from borosilicate glass nanocapillaries in ammonium acetate buffers with detergents (β-OG, C8E4). In-source activation, trap CID, pre- or post-IMS ECD, and single-pass cyclic IMS were performed. CCS values were calibrated using IMScal19 software with polyalanine and bovine serum albumin standards.

Used Instrumentation

• SELECT SERIES Cyclic IMS mass spectrometer
• Pre-mobility SID device (eMSion) and ExD cell
• Trap collision cell for CID
• Nano-electrospray capillaries
• Masslynx and waters_connect software
• IMScal19 calibration tool

Main Results and Discussion

Complex-down sequencing of the streptavidin tetramer achieved 78 % coverage by ECD and 89 % by CID, totaling 98 %. GroEL’s CCS was measured at 220 nm2, consistent with reference data. Membrane protein analysis showed effective micelle removal: OmpF (23+ charge) CCS was 6380 Å2 and pfMATE (10+ charge) CCS was 3655 Å2. Ion mobility separation of SID fragments resolved species sharing identical m/z but differing in shape and mass. Automated SID enabled rapid voltage ramps for optimal dissociation profiling.

Benefits and Practical Applications

• High-resolution separation of protein conformers and oligomers
• Comprehensive top-down sequencing through combined CID and ECD workflows
• Robust detergent micelle removal for intact membrane protein characterization
• Accurate CCS measurements with advanced calibration
• Automated SID enhances throughput and reduces manual intervention

Future Trends and Potential Applications

Routine integration of automated SID and high-resolution IMS in high-throughput settings is anticipated. Future directions include analysis of larger protein assemblies, lipid-bound complexes, and dynamic systems. Improved calibration standards, software tools, and coupling with computational modeling will further advance structural insights, with broad adoption in pharmaceutical and structural biology research.

Conclusion

The SELECT SERIES Cyclic IMS platform delivers an unrivalled suite of fragmentation modes (CID, ECD, SID), high-resolution ion mobility separation, and automated workflows, enabling detailed structural and sequencing studies of soluble and membrane proteins in native‐like states.

References

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