Advances in hardware design and function of the new timsOmni MS platform

Importance of the Topic

The comprehensive structural characterization of biomolecules, especially proteins and glycopeptides, is critical for applications in biopharmaceutical development, quality control and fundamental research. Advanced fragmentation techniques that preserve labile modifications, such as electron-based dissociation methods, are essential to obtain high-resolution sequence and structural information. The integration of ion mobility separation further enhances the ability to dissect complex mixtures and conformational states.

Goals and Overview of the Study

This work presents recent hardware and methodological developments implemented on the timsOmni MS platform, designed to combine trapped ion mobility spectrometry (TIMS) with multiple complementary fragmentation techniques including ECD, EID and CID. The study aims to demonstrate the platform’s versatility for soft fragmentation, dynamic ion accumulation and targeted glycopeptide analysis.

Used Instrumentation

• ESI sources (analytical, micro-flow VIP-HESI, nano-flow CaptiveSpray and offline NEOS) for broad flow-rate compatibility and native MS.
• 1.3 mm i.d. inlet capillary feeding ions into an RF ion funnel at 10 mbar.
• Stacked-ring RF ion guide for collisional activation (CIU, isCID) upstream of TIMS.
• Dual gated TIMS analyzer with mobility selection and high-capacity ion accumulation.
• Post-TIMS collisional activation region at 0.5 mbar.
• Quadrupole mass filter to 4500 m/z and Omnitrap cell for MSn and electron-based fragmentation (eXd).
• OA-TOF analyzer with mass-selective ejection and fast acquisition.

Methodology

The platform employs a dual-gate TIMS setup to separate ions by mobility before selective transmission for fragmentation. Electron capture dissociation (ECD) and electron-based dissociation (EID/EED) occur in the Omnitrap cell with fine control of electron energy. Collision-induced unfolding (CIU) combined with TIMS-ECD was used to probe protein conformations. Targeted analysis of low-abundance N-glycopeptides from transferrin digest was achieved using dynamic precursor accumulation and alternating ECD/EID scans.

Main Results and Discussion

CIU-TIMS-ECD experiments on carbonic anhydrase revealed distinct unfolding transitions and allowed mapping of flexible backbone regions. Dynamic accumulation in the Omnitrap improved signal-to-noise in MS2 spectra of Adalimumab subunits and transferrin glycopeptides. The combined ECD/EID approach provided complementary fragment coverage, enabling site-specific glycan localization on peptides and high sequence coverage (>75%). Chromatographic traces demonstrated robust detection of low-level glycopeptide species with accurate precursor isolation and fragmentation timing.

Benefits and Practical Applications

• Enhanced structural insights through complementary fragmentation methods in a single platform.
• Soft dissociation preserves labile modifications for glycoproteomics and native MS studies.
• High throughput via dynamic ion accumulation and rapid MS/MS cycles.
• Mobility separation adds conformational resolution and reduces spectral complexity.

Future Trends and Opportunities

Integration of advanced ion mobility with high-resolution MSn and electron-based fragmentation will drive deeper analysis of proteoforms, membrane proteins and large glycoprotein complexes. Further automation of mobility-directed workflows and real-time data-driven ion selection can increase throughput in biopharmaceutical QA/QC. Expanding accessible m/z range and improving eXd efficiency at low charge states will broaden applications in structural biology and metabolomics.

Conclusion

The timsOmni platform represents a versatile and powerful solution for structural and sequence characterization by uniting TIMS, CID, CIU, ECD and EID within one instrument. Its ability to selectively accumulate, separate and fragment ions with fine electron energy control offers superior data quality for complex biomolecular analyses.

References

• No reference list was provided in the original document.