Advanced Native and Top-Down MSⁿ Workflows on the timsOmni Platform Enable Deep Structural Analysis of Membrane Protein Complexes

Importance of the Topic

Membrane proteins are central to cellular physiology and represent major drug targets, but their structural characterization is complicated by heterogeneous oligomeric states, diverse proteoforms, and a variety of lipid and cofactor interactions. High-resolution native mass spectrometry combined with top-down MSn sequencing provides a route to identify proteoforms, localize modifications such as lipidation, and describe the architecture of intact membrane-protein assemblies. The work summarized here demonstrates how advanced MSn strategies on the timsOmni platform can resolve N-terminal lipid anchors on individual subunits within a multimeric membrane complex, addressing a critical analytical bottleneck in structural proteomics.

Objectives and Study Overview

The study aimed to develop and demonstrate a native top-down MSn workflow that:

• systematically disassembles an intact membrane-protein complex into subunits,
• enriches and sequences low-abundance lipidated fragment ions, and
• structurally localizes and elucidates N-terminal lipidation chemistries directly from protein-derived fragments.

As an exemplar, the heteropentameric BamABCDE β-barrel assembly machinery was interrogated to characterize N-terminal lipid anchors on its lipoprotein subunits.

Methods and Analytical Strategy

The workflow integrates native MS with staged fragmentation (isCID, psMS2 CID, resonance CID (RCID) and MSn up to MS4) and targeted ion enrichment to maximize signal for low-abundance, lipid-bearing fragments. Key methodological elements:

• Initial gentle dissociation of the intact BamABCDE assembly by in-source collision-induced dissociation (isCID) to produce intact subunit ions.
• Quadrupole selection of subunit charge states (m/z < 4500 Th) and use of the Omnitrap Q2 segment to isolate and accumulate larger ions for enhanced sensitivity.
• Application of MS3 RCID to generate low-abundance b-type fragments that retain lipid modifications.
• Selective isolation and cyclic ion enrichment of these modified fragment ions to improve signal-to-noise and permit further MS4 RCID fragmentation for structural elucidation.
• Data processing of MSn datasets using OmniScape for annotation and interpretation.

Additional experimental parameters included nanospray sample introduction via coated open emitters, ion acceleration for desolvation at 120–200 eV and operating pressures near 0.5 mbar when performing isCID and related steps.

Instrumentation Used

The analytical platform combined the following components and modes:

• timsOmni platform (Bruker) incorporating an Omnitrap module for Q2-based isolation and accumulation.
• Analytical quadrupole mass filter for precursor selection.
• Time-of-flight (TOF) mass analyzer for mass detection.
• nESI (nano-electrospray) sample introduction with coated open emitters and a NEOS-type source configuration.
• RCID (resonance CID) in the Omnitrap Q2 segment for MS3 and MS4 fragmentation.
• Software OmniScape for MSn data analysis and interpretation.

These elements were combined to enable isolation, enrichment cycles, and deep sequential fragmentation of lipid-carrying peptide backbone fragments.

Main Results and Discussion

The workflow delivered systematic dissociation of the intact BamABCDE complex and enabled targeted interrogation of individual subunits. Two subunits, BamE and BamD, were characterized in detail:

• MS3 RCID generated low-abundance b-type fragments that retained lipid modifications; selective isolation and enrichment of these fragments markedly improved signal-to-noise, enabling MS4 experiments.
• MS4 RCID of a lipid-bearing b12(2+) ion from BamE produced a comprehensive series of neutral-loss fragments consistent with palmitic acid (C16:0), oleic acid (C18:1), and a 1-oleoyl-2-palmitoyl glycerol species. Observed neutral losses also indicated an S-linked diacylglycerol on the N-terminal cysteine, and an additional hexadecyl loss supported linkage of a second palmitoyl moiety to the cysteine. Together, these fragment ions allowed unambiguous structural assignment of the lipid anchor architecture.
• MS3 and follow-up MSn analysis of BamD produced similar patterns of lipid-associated mass shifts and fragment neutral losses (including CH2 and C2H4 series), enabling confident localization of the lipid on the N-terminal cysteine of BamD as well.

These results illustrate that targeted ion enrichment plus staged RCID fragmentation can extract chemically interpretable lipidation signatures from low-abundance protein-derived fragments, directly revealing the composition and attachment chemistry of N-terminal lipid anchors within a multimeric membrane complex.

Benefits and Practical Applications

The presented approach offers several advantages for membrane-protein structural proteomics:

• Direct localization and structural elucidation of lipid modifications without reliance on separate lipidomics workflows or chemical derivatization.
• Capability to analyze low-abundance, labile modifications by targeted ion enrichment and accumulation, improving detection limits for modified fragments.
• Preservation of native assembly context during dissociation, enabling correlation of proteoform-level modifications with complex stoichiometry and subunit interactions.
• Compatibility with high-throughput MSn data processing via dedicated software (OmniScape), facilitating annotation and interpretation for proteomics and structural biology laboratories.

Practical use cases include mapping lipoprotein anchors, studying lipid–protein interactions in membrane complexes, and integrating modification localization into models of complex assembly and function in basic and translational research.

Future Trends and Applications

Expected developments and broader opportunities stemming from this work include:

• Extension of targeted enrichment MSn workflows to other classes of labile post-translational modifications (e.g., prenylation, glycosylphosphatidylinositol anchors) and to heterogeneous membrane complexes from native sources.
• Improved automation and higher-throughput MSn acquisition and interpretation, enabling routine incorporation of deep top-down lipidation mapping into proteomics pipelines.
• Integration with ion mobility separation on timsOmni-like platforms to add conformational context to modification localization and to separate isobaric proteoforms prior to MSn.
• Application in drug-discovery and quality-control workflows where precise mapping of membrane-protein modifications affects binding, stability, or function.

Conclusion

Advanced native top-down MSn workflows on the timsOmni platform, combining in-source dissociation, targeted quadrupole/Omnitrap isolation, resonance CID, and cyclic ion enrichment, enable confident localization and detailed structural characterization of N-terminal lipidation within multimeric membrane-protein complexes. The approach recovers chemically diagnostic neutral-loss patterns from low-abundance fragments and reveals attachment chemistries directly from protein-derived ions, providing a powerful tool for structural proteomics of membrane assemblies.

References

1. Jeremy Norris, Abraham O. Oluwole, Mariangela Kosmopoulou, Dodge Baluya, Ioanna Barla, Athanasios Smyrnakis, Dimitris Papanastasiou, Weston Struwe, Carol V. Robinson. Advanced Native and Top-Down MSn Workflows on the timsOmni Platform: Enable Deep Structural Analysis of Membrane Protein Complexes. ASMS 2026 – WP661. Bruker Scientific LLC and University of Oxford affiliations. 2026.