Novel ion activation workflows in the hybrid Orbitrap-Omnitrap platform empower top-down and bottom-up proteomics

Importance of the topic

Proteomic analysis provides critical insights into protein structure, function and post-translational modifications. The hybrid Orbitrap-Omnitrap platform unifies complementary ion activation workflows to expand sequence coverage and structural characterization in both top-down and bottom-up proteomics.

Objectives and Study Overview

This study aimed to integrate multiple ion activation methods into a single Omnitrap device coupled with high-resolution Orbitrap mass spectrometers. Novel multidimensional MSn workflows were developed to enhance peptide and intact protein analysis, leveraging a combination of electron-based, collisional and photodissociation techniques.

Methodology and Instrumentation

• Hybrid Omnitrap linear ion trap interfaced with Q Exactive HF™ and Exploris™ 480 Orbitrap mass spectrometers (Thermo Fisher Scientific) with Biopharma upgrade.
• Incorporated ion activation methods: ECD, EID, HCD, SHCID, vacuum-UV photodissociation (VUV-PD), hydrogen atom attachment (HAA) and ion-activated dissociation (IAD).
• Lossless ion transfer between the HCD collision cell and Omnitrap enabled MS2–MS4 workflows.
• Chromatographic separation: 25 cm PepSep C18 column in DDA mode; acquisition enhanced by FTMS Booster system.

Main Results and Discussion

• EID and ECD reactions performed within 50–80 ms improved sequence coverage for BSA peptides across all charge states compared to HCD alone.
• Unique multidimensional MSn workflows produced comprehensive fragmentation patterns of native ubiquitin, achieving up to 100% sequence coverage and enabling residue-level analysis.
• Combination of HAA and photodissociation delivered complementary ion series and distinguished isobaric residues such as leucine and isoleucine.
• MS3 CID of radical protein ions enhanced signal-to-noise and supported identification of labile modifications.

Benefits and Practical Applications

• Comprehensive mapping of protein primary structure and PTMs in single experiments.
• Rapid ExD methods compatible with chromatographic timescales accelerate bottom-up proteomics.
• Flexible targeting of specific peptide classes, modifications and charge states improves analytical depth.
• Applications in quality control, biopharmaceutical development and academic research requiring detailed proteomic analysis.

Future Trends and Potential Applications

• Integration with ion mobility separation and advanced LC platforms to further increase proteome coverage.
• Automation of multidimensional MS workflows for high-throughput analysis of complex biological samples.
• Expansion of activation chemistries (e.g., multi-wavelength UVPD) for enhanced PTM localization.
• Extension of workflows to metabolomics and glycoproteomics for detailed structural elucidation.

Conclusion

The hybrid Orbitrap-Omnitrap platform consolidates a broad spectrum of ion activation methods into a versatile analytical tool, delivering unprecedented sequence coverage and structural insights in top-down and bottom-up proteomics. This approach establishes a foundation for routine, high-resolution MS workflows in both fundamental research and applied settings.