Infrared Activation Enables Native Top-Down MS Analysis of Membrane Proteins and Protein Complexes

Importance of the Topic

Native analysis of membrane proteins and their complexes by mass spectrometry is essential for understanding their structural and functional roles in biological systems. Traditional collision-based approaches often struggle with efficient detergent removal and sequence coverage, while gentle activation can better preserve labile interactions and post-translational modifications.

Objectives and Study Overview

This study evaluates infrared-based activation methods for top-down mass spectrometry of membrane proteins. Key aims include:

• Developing infrared demicellization (IRD) to gently liberate proteins from detergent micelles.
• Comparing infrared multiphoton dissociation (IRMPD) and activated-ion electron transfer dissociation (AI-ETD) for sequence coverage.
• Demonstrating comprehensive analysis on model proteins AqpZ, AmtB, and the bacterial BAM complex.

Methodology and Instrumentation

Membrane proteins (AqpZ, AmtB, BAM complex) were expressed in E. coli, purified, and exchanged into ammonium acetate with detergent (C8E4 or DDM). Mass spectra were acquired on a Thermo Scientific Orbitrap Eclipse Tribrid modified with a 10.6 μm CO2 laser interfaced to the quadrupole linear ion trap (Q-LIT). Key steps:

• IRD: laser power and irradiation time were optimized to remove detergent while preserving noncovalent interactions.
• Fragmentation: IRMPD and AI-ETD were applied to IRD-liberated proteins at optimized parameters.
• Data analysis: TDValidator software with stringent tolerances was used to assign fragment ions.

Main Results and Discussion

IRD consistently freed intact protein complexes from detergents across a range of energies and durations, as shown for AqpZ and AmtB. IRMPD yielded abundant sequence-informative fragments at low Mathieu q values, overcoming limitations of collisional methods. AI-ETD provided complementary c/z ions with reduced non-dissociative ETnoD. Combining both techniques achieved near-complete coverage of AqpZ and substantial coverage of AmtB. Application to the 200 kDa BAM complex enabled assignment of subcomplex components and identification of N-terminal lipid modifications.

Benefits and Practical Applications

Infrared activation strategies offer:

• Precise control over detergent removal to preserve labile bindings.
• Enhanced sequence coverage for large, poorly charged membrane proteins.
• Capability to analyze multi-protein assemblies and detect post-translational modifications.

Future Trends and Applications

Advancements in ion isolation and fill strategies are expected to further boost fragment intensities and sequence coverage. Routine adoption of IR-based methods may enable high-throughput native top-down analysis of diverse membrane complexes, PTM mapping, and drug–protein interaction studies.

Conclusion

Infrared-driven demicellization, IRMPD, and AI-ETD constitute a powerful workflow for native top-down mass spectrometry of membrane proteins and complexes, achieving efficient detergent removal, robust fragmentation, and deep sequence characterization.

Reference

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