USING TRAVELING WAVE ION MOBILITY MASS SPECTROMETRY TO INVESTIGATE NATIVE AND DEUTERATED PROTEINS AND PEPTIDES – NEW CAPABILITIES FOR CORE LABS

Significance of the Topic

The analysis of peptides and proteins requires both rapid separations and high resolution to handle complex mixtures and preserve structural information. Traveling-wave cyclic ion mobility spectrometry (cIM) coupled with time-of-flight mass spectrometry addresses bottlenecks in liquid chromatography by providing an extra separation dimension. This capability is particularly important for hydrogen–deuterium exchange mass spectrometry (HDX-MS) workflows, where fast analysis is critical to maintain deuterium labeling patterns and reveal subtle conformational states.

Aim and Overview of the Study

This work explores the performance of cIM-enabled MS for:

• Rapid peptide separation under a 3-minute LC gradient.
• Direct infusion of complex peptide digests.
• Retention of backbone and sidechain deuteration during multi-pass IMS.
• Detection of hidden protein conformers via gas-phase separation.

Methodology and Used Instrumentation

Samples: Enolase tryptic digest (1 pmol), synthetic peptide P1, and intact myoglobin.
LC conditions: ACQUITY BEH C18 column (2.1×50 mm, 1.7 μm) at 45 °C, 1–90 % acetonitrile gradient over 3 min, 0.6 mL/min flow.
Infusion: 3 μL/min from 250 μL syringe for labeled and quenched samples.
Mobile phases: water/0.1 % formic acid (A), acetonitrile/0.1 % formic acid (B).
Instrument: SELECT SERIES Cyclic IMS–enabled quadrupole time-of-flight mass spectrometer.
Buffer gases: nitrogen in mobility and trap cells; helium in the cIM entrance.
Software: MassLynx v4.2 for acquisition; DriftScope for data analysis.

Main Results and Discussion

Multi-pass IMS resolution increased from ~65 to ~200 (Ω/ΔΩ) over 1–10 passes, effectively separating overlapping isotope clusters of deuterated peptides.
Rapid LC runs achieved 82 % sequence coverage of enolase within 3 min, comparable to longer gradients.
Backbone amide deuteration remained intact after up to ten cIM passes, confirming minimal gas-phase back-exchange for critical HDX sites.
Sidechain deuteriums exhibited progressive back-exchange within the cIM device, independent of trapping time, indicating that gas-phase ion-neutral reactions occur during cycling.
Direct infusion experiments resolved congested features around m/z 700 by distinguishing four overlapping species after five passes.
Analysis of myoglobin 16+ charge state from D2O solution revealed at least five distinct conformers after ten passes, uncovering structural heterogeneity not evident in H2O analysis.

Benefits and Practical Applications

• Accelerates proteomic workflows by reducing LC times without sacrificing peak capacity.
• Enables high-resolution separation of deuterated peptides for HDX-MS, preserving valuable structural labels.
• Facilitates infusion-based assays, streamlining sample throughput in core labs.
• Offers a platform for gas-phase ion–neutral chemistry studies, such as controlled back-exchange or covalent labeling.

Future Trends and Opportunities

Integration of cyclic IMS into routine proteomic and HDX-MS workflows to enhance throughput and structural insight.
Development of tailored gas-phase chemistries to probe labile side-chain interactions and post-translational modifications.
Application to larger protein complexes and intact assemblies for advanced structural biology and drug discovery.
Combining cIM data with machine-learning models to predict conformational ensembles from mobility and mass data.

Conclusion

Traveling-wave cyclic IMS-TOF mass spectrometry delivers rapid, high-resolution separations suitable for both LC and infusion analyses. The method preserves backbone deuteration while localizing sidechain back-exchange to the mobility device, enabling detailed HDX-MS experiments. Multi-pass cycling reveals hidden conformers of intact proteins, offering new avenues for structural proteomics in core laboratories.