Novel XL-MS analysis workflows for membrane protein characterization

Importance of the Topic

Membrane proteins constitute nearly 30% of the cellular proteome and represent over 60% of current drug targets. Their structural characterization is hindered by low expression yields, hydrophobicity, and the need for suitable membrane mimetics. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful approach for probing protein architecture and interactions but requires tailored workflows to address challenges posed by detergents, nanodiscs, or styrene–maleic acid lipid particles (SMALPs).

Goals and Study Overview

This study aimed to develop end-to-end XL-MS protocols for membrane proteins in different solubilizing environments. Key objectives included:

• Comparing in-gel and in-solution digestion workflows for crosslink analysis of a Salmonella enterica enzyme (WbaP1) in SMALPs.
• Evaluating three membrane-permeable crosslinkers (DSS, DSSO, tBuPhoX) for sequence coverage and crosslink depth.
• Establishing a microwave-assisted acid hydrolysis (MAAH) and FAIMS-enhanced LC-EThcD method for disulfide bond mapping of G protein-coupled receptors (AT2R).
• Applying optimized protocols to HIV restriction factor SERINC3 to improve structural resolution of dynamic regions.

Methodology and Instrumentation

• Crosslinking reagents: DSS, DSSO, tBuPhoX applied to proteins in detergent (DDM/CHS, LMNG) or SMALP preparations.
• Digestion strategies: in-gel vs in-solution using trypsin and pepsin; TiO2 enrichment for phospho-crosslinker (tBuPhoX) samples.
• Disulfide mapping: MAAH in original buffers followed by C18 desalting without buffer exchange, combined with FAIMS-LC-EThcD MS/MS.
• Native MS: online buffer exchange (OBE) with DMT on Q Exactive UHMR for intact mass and oligomeric state analysis.
• Instrumentation: Orbitrap Ascend Tribrid MS with/without FAIMS Pro Duo; Vanquish Neo UHPLC with EASY-Spray column; data processed by Proteome Discoverer 3.2 and XlinkX 3.2; visualization via XMAS in ChimeraX and PyMOL.

Main Results and Discussion

1. Sequence coverage: In-solution workflows achieved >90% coverage for WbaP1 versus ~82% in-gel, with more crosslinked spectral matches for both DSS and tBuPhoX.
2. Crosslink distribution: DSS yielded the highest overall crosslinks; tBuPhoX enriched crosslinks at membrane-adjacent dimer interfaces.
3. Disulfide mapping: MAAH-FAIMS-LC-EThcD confirmed two expected S–S bonds and three surface cysteines in AT2R, achieving >80% coverage without buffer exchange.
4. SERINC3 analysis: Native MS+DMT characterized oligomeric state in DDM/CHS and LMNG; XL-MS plus MAAH highlighted flexible H8 domain interactions between helical bundles, complementing cryo-EM data.

Practical Benefits and Applications

These optimized workflows enable detailed structural mapping of membrane proteins in near-native environments. Key advantages include:

• High sequence coverage (>80–95%) and extensive crosslink identification for topology and interaction studies.
• Compatibility with detergents and SMALPs, reducing sample manipulation and preserving native conformations.
• Streamlined disulfide bond mapping without buffer exchange, accelerating analysis of cysteine-rich receptors.
• Integration of native MS for mass and stoichiometry assessment, supporting a multi-modal structural characterization pipeline.

Future Trends and Opportunities

Continued development of cleavable and enrichment-friendly crosslinkers will further enhance membrane protein analysis. Emerging directions include:

• Combining XL-MS with cryo-EM or cryo-ET to refine dynamic regions unresolved by imaging alone.
• Expanding MAAH protocols for other post-translational modifications and complex membrane assemblies.
• AI-driven modeling tools integrated with XL-MS datasets to predict conformational landscapes.
• High-throughput automation of native MS-DMT workflows for rapid screening of membrane protein libraries.

Conclusion

This work delivers robust, end-to-end XL-MS workflows tailored for membrane proteins in diverse mimetics. By combining optimized digestion, crosslink chemistries, FAIMS-enhanced acquisition, and native MS, researchers can achieve comprehensive mapping of protein architecture, disulfide networks, and dynamic interfaces. These methods provide a versatile platform for drug target validation, receptor biology, and membrane complex characterization.

Instrumental Details

• Mass spectrometers: Thermo Scientific Orbitrap Ascend Tribrid; Q Exactive UHMR with OBE-DMT.
• LC system: Thermo Scientific Vanquish Neo coupled to EASY-Spray 75 μm × 25 cm column.
• FAIMS Pro Duo interface for gas-phase ion separation.
• Data software: Proteome Discoverer 3.2 (XlinkX node 3.2, SEQUEST HT); ChimeraX XMAS plugin; PyMOL.

References

1. Dodge GJ et al. Mapping the initiating phosphoglycosyl transferase from S. enterica O-antigen biosynthesis in liponanoparticles. eLife. 2024;12:RP91125.
2. Heissel S et al. Fast and Accurate Disulfide Bridge Detection. Mol Cell Proteomics. 2024;23(5):100759.
3. Liu W et al. Automated Membrane Protein Analysis by Native Mass Spectrometry. Anal Chem. 2023;95(47):17212–17219.
4. Lagerwaard IM et al. XMAS – Integrative Modeling in ChimeraX. bioRxiv. 2022.
5. Zhang H et al. Structural basis for selectivity and diversity in angiotensin II receptors. Nature. 2017;544(7650):327–332.
6. Leonhardt SA et al. Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry. Nat Commun. 2023;14(1):4368.