Offline tandem MSn workflows on the timsOmni platform for deep sequencing of intact proteins and mAbs

Importance of the topic

The application of top-down mass spectrometry (TDMS) to intact proteins and monoclonal antibodies (mAbs) promises detailed sequence information and disulfide bond characterization. However, challenges such as signal dilution across multiple charge states, overlapping isotopic envelopes, and limited sequence coverage have impeded broad adoption in structural and quality control workflows.

Objectives and study overview

This work demonstrates novel offline tandem MSn workflows on the timsOmni platform to enhance deep sequencing of non‐reduced intact mAbs. The study aims to integrate in‐source collision‐induced dissociation (isCID) with multiple-stage activations (ECD, EID, RCID) to achieve comprehensive sequence mapping and disulfide bond analysis.

Methodology

• Sample preparation: NISTmAb diluted to 3.3 µM in 50:50 H2O/ACN with 0.1% formic acid.
• Electrospray ionization: NEOS nanoESI source at ~1.2 kV.
• Offline MSn: isCID for subunit release, followed by MS2 CID, MS3 ECD/EID, and MS4 RCID in the Omnitrap cell.
• Data acquisition: Extended scan averaging across charge states (e.g., 49+, 52+, 55+), with reaction times of 50 ms and accumulation of products for ~1 s.
• Data analysis: OmniScape software for sequence mapping and fragment annotation.

Instrumentation

• timsOmni mass spectrometer with integrated Omnitrap section.
• High‐capacity orthogonal acceleration time‐of‐flight (OA‐TOF) analyzer (>5×10⁶ charges/ms).
• NEOS nanoESI ion source and coated nanospray tips.
• Quadrupole mass filters for precursor and fragment selection.

Main results and discussion

• MS2 CID generated abundant b‐type ions, which were selectively processed through MS3 ECD/EID and MS4 RCID stages.
• Combined multimodal fragmentation yielded sequence coverages up to 88% for heavy‐chain fragments and over 80% for light chain regions.
• ps.MS3 ECD of light chain 10+ precursor delivered near‐complete coverage outside intrachain disulfide loops; subsequent RCID of charge‐reduced ions achieved full reduction of the first disulfide bond.
• Partial reduction of intrachain bonds in heavy‐chain fragments indicated differential accessibility and highlighted the method’s capability for disulfide mapping.

Benefits and practical applications

• Enhanced sequence coverage for intact, non‐reduced mAbs supports detailed structural characterization.
• Capability for targeted disulfide bond analysis informs biotherapeutic quality control and comparability studies.
• Offline MSn workflows increase flexibility for complex sample types and extended scan durations.

Future trends and potential applications

• Integration of timsOmni MSn approaches into automated QC pipelines for biologics manufacturing.
• Expansion to native TDMS for intact protein complexes and multi‐subunit assemblies.
• Development of real‐time data processing algorithms for rapid fragment annotation and decision support.

Conclusion

Offline isCID‐MSn methodologies on the timsOmni platform provide a powerful strategy for deep sequencing and disulfide bond mapping of intact non‐reduced mAbs. The combination of complementary fragmentation modes and high‐capacity instrumentation addresses key limitations of conventional TDMS, paving the way for robust structural analyses in research and industrial settings.

Reference

No references provided in the original document.