Location, Location, Location… Native Top-Down Mass Spectrometry Provides Binding Site Information for Protein-Ligand and Protein- Protein Interactions

Význam tématu

Native top-down mass spectrometry (nTDMS) is an emerging analytical approach that simultaneously provides intact mass, stoichiometry and localized sequence/structural information for noncovalent protein complexes. The ability to fragment gas-phase complexes while preserving labile noncovalent interactions enables direct mapping of ligand binding sites and protein–protein interfaces without requiring prior digestion or extensive sample manipulation. This capability addresses critical needs in structural proteomics, biopharmaceutical characterization, and mechanistic studies of macromolecular assemblies.

Cíle a přehled studie / článku

The study demonstrates how trapped electron-driven dissociation (trapped EXD, encompassing ECD and EID) implemented on the timsOmni MS platform, combined with multistage MSn workflows, can define ligand contact residues and subunit interaction regions in native protein complexes. Key model systems are holo-myoglobin and L‑malate dehydrogenase (L‑MDH) homodimer; the work shows sequencing depth, retention of noncovalent ligands on fragments, and localization of protected interface stretches.

Použitá metodika a instrumentace

Experimental design and workflow:

• Samples: commercially available holo-myoglobin (horse) and L‑MDH (pig) buffer exchanged into 150 mM ammonium acetate and electrosprayed at ~5 μM.
• Ionization and pre-activation: nano-ESI using Humanix Cellomics emitters with the NEOS source; in-source CID (isCID) in argon to aid desolvation and to release subunits.
• MSn workflow: m/z selection (RF isolation up to 4500 Th), accumulation in the Bruker Omnitrap (Q5) and application of resonance-excitation CID (RCID), electron capture dissociation (ECD), electron ionization dissociation (EID) and combined modes (including ECciD) across MS2 and MS3 stages.
• Acquisition strategy: long ion accumulation (>100 ms) and averaging over ~3 minutes to improve signal-to-noise and enable deep sequencing coverage.
• Data analysis: OmniScape software for top-down interpretation and mapping.

Použitá instrumentace

• timsOmni MS platform with external electron source coupled to Q5 (Omnitrap).
• NEOS electrospray source with Humanix Cellomics emitters.
• OmniScape software for MSn top-down data processing.

Hlavní výsledky a diskuse

L‑Malate dehydrogenase (L‑MDH):

• MS2 RCID of the 17+ L‑MDH dimer (≈66 kDa) produced 44% sequence coverage (SC), with predominant y‑ions from the solvent‑exposed C‑terminus consistent with structure.
• MS2 ECD and MS2 EID of the 17+ dimer each gave ~49% SC, also biased to the C‑terminus.
• MS3 experiments on the monomer (released by isCID) — especially MS3 EID — penetrated further into the monomer core and achieved up to 63% SC for the monomer.
• Combining MS2 data from both monomer (z=10+) and dimer (z=17+) produced a cumulative SC of 92%.
• Notably, residues M227–R233 yielded no MS2 fragments, identifying this stretch as protected and likely forming a tight protein–protein interface in the dimer.

Holo‑myoglobin (holo‑Mb):

• MS2 ECD of 8+ holo‑Mb generated c/z ions between H64 and H93, observed both with and without bound heme, indicating that heme can remain attached to product ions and may bind to either coordinating histidine after dissociation.
• MS2 RCID produced 68% SC and commonly resulted in heme loss; observed heme fragments and intact heme losses matched expected porphyrin fragmentation patterns (losses of carboxymethyl and methyl substituents).
• ECD produced charge‑reduced radical intact holo‑Mb; subsequent MS3 CID of the 7+ radical yielded fragments in the H64–H93 region but also displayed heme loss that depended on fragment size—larger fragments more often retained heme.
• Many heavy c‑type fragments showed broadened isotopic envelopes shifted by +2 H, interpreted as evidence for altered iron/porphyrin oxidation states after collisional activation.
• Combined fragmentation data delivered overall SC ≈99% and demonstrated retention of heme on fragments localized to either coordinating histidine.

Přínosy a praktické využití metody

• Native top‑down MS with trapped EXD enables direct localization of ligand contact residues and mapping of subunit interfaces without denaturation or proteolysis.
• Retention of noncovalent ligands on fragment ions improves confidence in assigning binding sites and in distinguishing between alternative binding residues (e.g., two histidines in myoglobin).
• Multimodal fragmentation (CID, ECD, EID and combined modes) and MSn workflows increase sequence coverage and allow complementary access to both termini and core regions of subunits.
• Applications include structural proteomics, characterization of therapeutic protein assemblies, mapping of post‑translational modifications in situ, and interrogation of macromolecular stoichiometry and dynamics.

Budoucí trendy a možnosti využití

• Integration with ion mobility and enhanced separation prior to MSn to better resolve conformers and coexisting complex stoichiometries.
• Higher‑energy control and optimized trapped electron parameters to extend identification of fragile interfaces and increase fragment yields for very large complexes.
• Automation of multistage workflows and improved software pipelines (e.g., machine‑assisted assignment) to accelerate mapping of binding sites and interfaces at scale.
• Combining nTDMS data with computational docking, molecular dynamics and cryo‑EM/X‑ray models to create hybrid structural models with residue‑level contact information.
• Extension of the approach to increasingly large and heterogeneous assemblies (membrane complexes, ribonucleoproteins) and to quantitative studies of ligand occupancy and binding kinetics.

Závěr

The work demonstrates that trapped EXD on the timsOmni platform, when combined with complementary CID modes and multistage MSn, provides a powerful and practical toolkit for native top‑down characterization of protein–ligand and protein–protein interactions. High sequence coverage, preservation of noncovalent ligands on product ions, and identification of protected interface regions exemplify how nTDMS can deliver residue‑level binding site information relevant for structural biology and applied biopharma analytics.

Reference

Data and findings were presented by Smyrnakis A., Kosmopoulou M.‑A., Papanastasiou D., Sakallioglu I., Greig M., Kensil M., and Loo J. A. as part of an ASMS 2026 communication reporting development and application of Bruker's timsOmni MS platform. The authors disclosed employment relationships with the instrument manufacturer.