Establishing a Decision Tree for Native Mass Spectrometry Analysis of Membrane Proteins in Complex Membrane Mimetics

Significance of the Topic

Membrane proteins represent the majority of drug targets and play central roles in cellular signaling and transport. Native mass spectrometry (nMS) offers unique insights into protein stoichiometry, assembly state and lipid interactions under near-physiological conditions. However, the diversity of membrane mimetics—detergent micelles, nanodiscs and styrene maleic-acid lipid particles (SMALPs)—presents challenges for routine nMS analysis. Establishing a structured workflow is essential to streamline characterization and accelerate discoveries in structural biology and pharmaceutical research.

Objectives and Study Overview

This work aimed to develop a decision tree guiding the selection of nMS approaches for membrane proteins in different mimetic environments. Key aims included:

• Rapid screening of protein–mimetic compatibility using online buffer exchange nMS (OBE-nMS).
• Resolution of heterogeneous complexes by direct infusion mass spectrometry (Direct Mass Technology mode).
• Charge‐state simplification for SMALP‐encapsulated proteins via data‐independent acquisition proton transfer charge reduction (DIA-PTCR).

Methodology

A multi‐step workflow was established:

• OBE-nMS screening: Thermo Scientific Vanquish Flex UHPLC with NativePac OBE-1 column and 200 mM ammonium acetate mobile phase, with or without detergents (C8E4, DDM, LDAO).
• Direct Mass Technology mode: direct infusion on a Q Exactive UHMR with Nanospray Flex ion source for high‐mass targets (> 100 kDa).
• DIA-PTCR: applied on an Orbitrap Ascend Tribrid instrument to reduce charge complexity for proteins < 100 kDa, revealing hidden proteoforms and lipid adducts.

Used Instrumentation

• Thermo Scientific Vanquish Flex UHPLC system
• NativePac OBE-1 online buffer exchange column
• Thermo Scientific Q Exactive UHMR Hybrid Quadrupole-Orbitrap mass spectrometer
• Thermo Scientific Orbitrap Ascend Tribrid Mass Spectrometer
• Thermo Scientific Nanospray Flex ion source

Main Results and Discussion

Detergent micelles

• OBE-nMS in 200 mM ammonium acetate with LDAO provided rapid detection and moderate charge reduction of GPCRs and other MPs.
• C8E4 caused aggregation or signal loss for sensitive receptors, while DDM maintained compatibility with some background noise.

Nanodiscs

• OBE-nMS preserved intact nanodisc assemblies when using detergent-free 200 mM ammonium acetate, generating broad m/z envelopes resolved by direct deconvolution.
• Direct Mass Technology confirmed molecular weights of protein–nanodisc complexes (~240 kDa) correlating with SEC-MALS.

SMALPs

• DIA-PTCR successfully simplified overlapping charge states and revealed proteoforms and lipid populations within SMALP particles.
• Sliding window deconvolution uncovered mass differences (700–750 Da) consistent with individual lipid molecules.

Decision Tree

1. Perform OBE-nMS as first screen with 200 mM ammonium acetate and LDAO for detergents or no detergent for nanodiscs.
2. If charge states are unresolved:
   • Use Direct Mass Technology mode for complexes > 100 kDa.
   • Apply DIA-PTCR for targets < 100 kDa.
3. For SMALPs, follow the same branching based on molecular weight.

Benefits and Practical Applications

• High-throughput screening: OBE-nMS completes compatibility checks in under 3 minutes per sample.
• Comprehensive coverage: combination of methods addresses a wide mass range and complex assemblies.
• Enhanced structural insights: detection of lipid binding, subunit stoichiometry, and assembly heterogeneity supports drug design and quality control in biotechnology.

Future Trends and Potential Applications

Continued advances in nMS instrumentation will expand mass range, resolution and speed. Integration with microfluidic sample handling and AI-driven decision support could automate method selection. Coupling native MS with complementary structural tools (cryo-EM, HDX-MS) will provide multi-scale insights into membrane protein dynamics and ligand interactions.

Conclusion

This study presents a clear decision tree for native MS analysis of membrane proteins across three mimetic systems. By combining online buffer exchange, direct infusion and proton transfer charge reduction, researchers can rapidly tailor nMS workflows to complex targets, improving throughput and data quality in structural biology and pharmaceutical pipelines.