Characterization of Endogenous Ligands in Membrane Proteins Using Dynamic Field Declustering (DFD) on the Cyclic™ IMS P20 Mass Spectrometer

Significance of the topic

Native mass spectrometry (MS) of membrane proteins provides direct readout of non-covalent complexes and endogenous ligands, critical for drug discovery and structural biology. Membrane proteins constitute a large fraction of drug targets but are difficult to analyse by native MS because detergents and membrane mimetics needed for solubility obscure mass measurement and can prevent isolation and fragmentation of bound ligands. The development of reliable gas-phase declustering and integrated ion mobility workflows addresses these limitations and improves confidence in ligand identification from biologically relevant preparations.

Objectives and overview of the study

• Demonstrate Dynamic Field Declustering (DFD) implemented on the Cyclic IMS P20 Mass Spectrometer effectively removes detergent micelles from membrane proteins in the gas phase while preserving endogenous lipid binders.
• Apply a pseudo-MS4 workflow—DFD release, quadrupole isolation, collision-induced release of ligand, ion mobility separation and post-IMS CID—to obtain fragment-level structural confirmation of endogenous lipids bound to bacteriorhodopsin (bR).
• Show how time-aligned ion mobility and fragmentation increase confidence in binder assignment from native-like membrane protein samples.

Methodology

• Sample: Native purple membrane containing bacteriorhodopsin (bR) prepared in 40 mM β-D-glucopyranoside (β-OG; ~2× CMC) with 200 mM ammonium acetate. Clarified and buffer-exchanged; estimated monomer concentration 1–5 µM.
• Ionization: Static nanospray from borosilicate capillaries with nanospray adaptor; positive polarity native electrospray.
• DFD operation: Sustained oscillating RF field applied within the StepWave™ ion guide to disrupt detergent-protein interactions and decluster micelles while retaining non-covalent lipid binders.
• Pseudo-MS4 workflow: (1) DFD to liberate protein from micelle, (2) quadrupole isolation of selected charged protein-ligand complex (e.g., [bR+PGP-ME]9+), (3) trap cell CID to dissociate the ligand, (4) Cyclic ion mobility separation to separate released ligand fragments from protein ions and other background, (5) post-IMS CID (transfer cell) to generate time-aligned product ions for structural assignment.
• Data processing: Spectral deconvolution using BayesSpray algorithm in waters_connect software; time-aligned mobility-resolved fragmentation to attribute fragments to specific precursors.

Used instrumentation

• Waters Cyclic IMS P20 Mass Spectrometer with DFD implemented in the StepWave ion guide.
• NanoLockspray II ionization source with static nanospray adaptor and borosilicate nanocapillaries.
• Key method settings reported: DFD frequency 60 kHz, DFD amplitude 175 Vpp; cone voltage 50 V; source temperature 100 °C; capillary voltage ~1.2 kV. Pseudo-MS4 collision settings: trap collision 100 V, transfer collision 30 V. Mobility single-pass T-wave mode (T-wave amp 22 V, velocity 375 ms−1) with short injection and separation times to preserve native complexes.

Results and discussion

• DFD efficiently removed detergent signal: Without DFD spectra were dominated by β-OG cluster ions with no visible protein. Activating DFD exposed high-quality protein charge states and ligand-bound complexes.
• Accurate intact mass measurement: Deconvolution returned an average bR mass of 27049.9 Da (4 ppm error relative to theoretical 27050.0 Da), demonstrating preservation of the protein and high mass accuracy after micelle removal.
• Detection of endogenous lipids: Deconvoluted peaks indicated sequential binding of up to three copies of an archaeal phospholipid denoted PGP-ME (+901 Da per copy) and binding of S-TGA-1 (+1220 Da per copy). Mixed stoichiometries (e.g., 1:1:1, 1:2:1 ratios of bR:PGP-ME:S-TGA-1) were observed, showing heterogeneous endogenous ligand populations.
• Pseudo-MS4 enabled structural confirmation: Selecting [bR+PGP-ME]9+ and applying trap CID released the lipid (observed at m/z 901.6). Ion mobility separated low m/z lipid-derived ions from protein charge states. Time-aligned post-IMS CID produced a clean product ion spectrum attributed to PGP-ME.
• Fragment assignments: Time-aligned fragments supported headgroup and fatty-acyl chain identification (headgroup ~248.99 m/z; fatty-acid related fragments at ~337.34, 339.36, 355.36, 357.37 m/z) and alkyl-chain substructure. Ions at 653 and 315 m/z were shown by mobility to be unrelated interferences, illustrating the power of mobility alignment to avoid misassignment.
• Mechanistic insight into dissociation: Observation of abundant [bR]9+ alongside residual [bR+PGP-ME]9+ and [bR]8+ indicates both neutral-loss and charged-loss dissociation pathways for the lipid, consistent with mixed gas-phase dissociation behaviour of lipid–protein complexes.

Benefits and practical applications

• DFD in the StepWave ion guide enables robust removal of detergents without requiring excessive collisional activation that could disrupt non-covalent ligands—critical for analysis of membrane proteins and their endogenous binders.
• The combined workflow (DFD + quadrupole isolation + cyclic ion mobility + post-IMS CID) permits pseudo-MS4 analysis that yields fragment-level structural confirmation of bound small molecules and lipids directly from native preparations.
• Applications include direct identification of endogenous ligands, support for screening campaigns (catch-and-release or affinity assays), and characterization of heterogeneous lipid binding to membrane proteins relevant to functional and pharmacological studies.

Future trends and applications

• Extension to larger and more complex membrane protein assemblies, including GPCR oligomers and ion channels, where retention of labile ligands during declustering is essential.
• Integration with high-throughput native-MS screening workflows to identify endogenous modulators or off-target binders from complex biological extracts.
• Improved analytical pipelines combining ion mobility-resolved fragmentation, advanced deconvolution algorithms and database-driven identification to accelerate confident annotation of unknown endogenous ligands.
• Potential adoption of tailored DFD and mobility parameters for different classes of membrane mimetics (nanodiscs, amphipols) to broaden applicability across membrane protein sample types.

Conclusion

Dynamic Field Declustering implemented on the Cyclic IMS P20 enables effective gas-phase removal of detergent micelles while preserving endogenous lipid binders, permitting high-accuracy mass measurement of membrane proteins. When combined with quadrupole isolation, cyclic ion mobility separation and post-IMS CID in a pseudo-MS4 workflow, this platform delivers fragment-level structural information that markedly increases confidence in ligand assignment from native membrane protein samples. The approach therefore strengthens native-MS as a tool for studying membrane protein–ligand interactions and supports applications in drug discovery and biochemical research.

Reference

1. Fiorentino F, Rotili D, Mai A. Native mass spectrometry-directed drug discovery: Recent advances in investigating protein function and modulation. Drug Discov Today. 2023;28:103548.
2. Hopper JTS. Mass Spectrometry: Application to Protein-Ligand Interactions. Encyclopedia of Biophysics. 2021:1–7.
3. El-Hawiet A, Shoemaker GK, Daneshfar R, Kitova EN, Klassen JS. Applications of a Catch and Release Electrospray Ionization Mass Spectrometry Assay for Carbohydrate Library Screening. Anal Chem. 2011;84:50–58.
4. Gault J, et al. Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins. Nat Methods. 2020;17:505–508.
5. Errey JC, Fiez-Vandal C. Production of membrane proteins in industry: The example of GPCRs. Protein Expr Purif. 2020;169:105569.
6. Sokratous K, et al. Enhanced Declustering Enables Native Top-Down Analysis of Membrane Protein Complexes using Ion-Mobility Time-Aligned Fragmentation. J Am Soc Mass Spectrom. 2024;35:1891–1901.
7. Le J, Loo JA. Native Top-Down Mass Spectrometry Characterization of Model Integral Membrane Protein Bacteriorhodopsin. J Am Soc Mass Spectrom. 2025;36:961–968.