Studying protein and peptide non-covalent complexes with ion mobility and electron capture dissociation mass spectrometry

Importance of the topic

Understanding non-covalent oligomerization of peptides and proteins is critical for both basic biochemistry and biopharmaceutical development. Oligomeric assemblies can modulate biological activity, contribute to disease pathology, and appear as problematic impurities in therapeutic formulations. Native-compatible mass spectrometry approaches that combine ion mobility separation and electron-based dissociation provide rapid, structurally informative readouts of oligomer stoichiometry, conformational families, and sequence-level information. This work demonstrates how ion mobility–QTOF MS with gentle activation and ECD can be applied to characterize higher-order oligomers of ubiquitin and the therapeutic peptide liraglutide, informing formulation and analytical strategies in drug development.

Objectives and study overview

The study aimed to:

• Characterize formation and structural features of non-covalent oligomers of ubiquitin (two truncation variants) and liraglutide under native and denaturing conditions.
• Use ion mobility (IM) to separate isobaric/isomeric oligomeric species and quadrupole isolation to gently dissociate complexes to determine stoichiometry.
• Assess collision cross section (CCS) trends across charge states and compare ECD sequence coverage for monomers versus oligomers.

The approach combined static nanospray native-like ionization, IM separation in a drift tube, quadrupole isolation with low collisional activation, and in selected experiments electron capture dissociation (ECD) using an ExD cell modification of the instrument.

Methodology

Samples and preparation: ubiquitin 1-74 (Sigma) and 1-76 (Bio-Rad) and liraglutide (Sigma) were studied without further purification. Oligomerization was induced from native buffers (20 or 100 mM ammonium acetate) and denaturing solvent (15% acetonitrile, 0.1% formic acid) at sample concentrations between 10 and 100 µM. Liraglutide samples were additionally incubated overnight at 35 °C to promote assembly.

Data acquisition and processing: static nanospray ionization with a low-temperature drying gas (room temperature, 1.5 L/min) preserved non-covalent assemblies during transfer. Mass spectra were acquired across m/z 100–20,000. Low-level quadrupole collisional activation was applied to fragment oligomers into smaller complexes and monomers for compositional analysis. Ion mobility drift times and CCS values were extracted and visualized; software used included Agilent MassHunter Acquisition v11.0, MassHunter Qualitative Analysis v12.0, ExDViewer v4.6.31 for fragmentation analysis, and Agilent IM-Browser 10.0 for IM processing.

Used instrumentation

• Agilent 6560 Ion Mobility Q-TOF mass spectrometer with drift-tube IM (nitrogen drift gas).
• ExD cell modification enabling electron capture dissociation (ECD) on the 6560 platform.
• Static nanospray ionization source; low drying-gas temperature and 1.5 L/min flow to retain oligomers.
• Quadrupole mass filter for precursor isolation and gentle CID-type activation.

Main results and discussion

• Retention of oligomers during ionization: Using room-temperature (low) drying gas preserved multiple oligomeric species in the gas phase for both ubiquitin and liraglutide. MS1 showed surviving multimers across a range of charge states.
• Effect of solvent and concentration on ubiquitin oligomerization: Denatured ubiquitin produced higher oligomerization levels than native ubiquitin. Increasing analyte concentration increased the extent of oligomer formation. Native ubiquitin favored more compact structures that limited higher-order association compared with denatured, more elongated forms.
• Fragmentation behavior and drift-time coincidence: Quadrupole isolation followed by gentle collisional activation was sufficient to dissociate multimers into defined smaller oligomers and monomers. Observed fragment ions maintained the same drift time as their precursor feature, consistent with direct dissociation of a specific conformational family.
• CCS trends for monomers versus dimers: For ions selected to avoid spectral overlap (odd charge states), CCS increased with charge state for both monomeric and dimeric ubiquitin species. Higher charge states corresponded to more elongated conformations; at comparable drift times lower-charge-state monomers and dimers had similar CCS as predicted by ion mobility theory, but at higher charge states dimers exhibited larger CCS than monomers due to longer drift times and more extended structures.
• Liraglutide oligomerization: Liraglutide formed extensive oligomers under the applied native-like conditions (including overnight 35 °C incubation). MS1 and IM separation revealed multiple charge-state distributions for oligomers, with monomer charge states up to 4+. Using IM selection and MS2, the authors determined the maximum oligomerization level observed under their conditions to be n = 17 (identified by fragmenting [n]n+ precursors into smaller components and monomers).
• Resolving isobaric/isomeric precursors: For even charge-state precursors where multiple species (e.g., monomeric + dimeric forms) can share m/z, ion mobility selection was critical to assign observed fragments unambiguously to specific precursor populations. Integrated MS2 without IM selection produced ambiguous fragment assignments, whereas IM-resolved MS2 spectra identified which mobility-resolved precursor produced each fragment.
• ECD performance: ECD on monomeric ubiquitin and liraglutide yielded expected sequence-informative fragmentation. ECD of intact oligomers proved challenging because isolation often triggered dissociation into lower-order oligomers, complicating spectral interpretation and reducing the quality of ECD sequence coverage for intact assemblies.

Benefits and practical applications of the method

• Combination of drift-tube IM and quadrupole isolation provides stoichiometric and conformational resolution of non-covalent oligomers that is difficult to obtain by MS1 alone.
• Low-temperature drying gas and gentle activation strategies allow preservation and controlled dissociation of labile assemblies, enabling composition analysis without complete unfolding.
• CCS measurements add structural constraints to interpret charge-state-dependent conformational changes and to distinguish compact versus elongated populations.
• The workflow is directly applicable to biopharmaceutical QA/QC for detecting and characterizing peptide/protein aggregates, guiding formulation optimization and stability testing.

Future trends and possibilities for application

• Instrumentation: Improvements in IM resolution and extended-mass-range ECD/ETD implementations will enable deeper interrogation of larger oligomers and heterogeneous assemblies without inducing dissociation during isolation.
• Method integration: Coupling native IM-MS with orthogonal separation (e.g., native SEC–MS), hydrogen/deuterium exchange, or cryogenic techniques could preserve solution-like conformations and enhance structural interpretation.
• Data analysis: Advanced deconvolution and modeling that integrate CCS, charge-state distributions, and fragment maps can improve assignment of heterogeneous mixtures and provide better stoichiometry/architecture models.
• Applications: Routine deployment in formulation screening, comparability studies, forced-degradation testing, and early-stage aggregation risk assessment for peptide and protein therapeutics.

Conclusions

This study demonstrates the utility of drift-tube IM–QTOF MS combined with gentle quadrupole isolation and selective ECD for characterizing non-covalent peptide and protein oligomers. Denaturing conditions and higher concentrations promoted ubiquitin oligomerization and yielded more extended conformations, whereas native ubiquitin adopted compact states that limited higher-order assembly. Liraglutide readily formed high-order oligomers (up to n = 17 under the tested conditions). Ion mobility separation was essential to resolve isobaric precursors and to attribute fragment ions correctly. ECD provided clear sequence information for monomers but was less informative for intact oligomers because of dissociation during isolation. Overall, the workflow offers a practical analytical platform to assess oligomer stoichiometry, conformational heterogeneity, and sequence identity relevant to therapeutic peptide and protein development.

References

• Yury V. Vasil’ev, Rachel Franklin. Studying protein and peptide non-covalent complexes with ion mobility and electron capture dissociation mass spectrometry. ASMS 2026, Poster TP 388. Agilent Technologies, Inc., Corvallis, OR, USA. Published June 30, 2026.