Comprehensive sequencing of cyclic peptides by top-down MS on a timsOmniplatform using next-generation fragmentation and OmniScapeprocessing

Importance of the topic

Cyclic peptides are increasingly important molecular entities in natural products, pharmaceutical discovery and industrial applications because they combine high target affinity and specificity with enhanced proteolytic stability and membrane permeability compared with linear peptides. Advances in synthetic chemistry have enabled large libraries of cyclic macrocycles to be produced rapidly, creating a demand for analytical workflows that can rapidly and unambiguously determine sequence, ring topology, post‑translational modifications (PTMs) and fragmentation behavior. High-confidence structural characterization of cyclic peptides is critical for drug discovery, quality control, and structure–activity relationship studies.

Objectives and study overview

This study demonstrates a comprehensive top-down mass spectrometry workflow for sequencing cyclic peptides using the Bruker timsOmni platform combined with OmniScape (OSc) data processing. The goals were to show how combined ion mobility separation, advanced fragmentation in an Omnitrap cell and exhaustive computational linearization enable unambiguous linear sequence assignment and detailed fragment-spectrum explanation, using cyclosporin A and related cyclic peptide examples to illustrate performance.

Instrumentation used

• Bruker timsOmni IMS Q‑Omnitrap ToF mass spectrometer (tims cartridge for trapped ion mobility separation and a ToF analyzer for detection).
• Omnitrap cell with integrated electron source enabling trapped electron‑based activation and multiple collision regimes.
• Ion sources: CaptiveSpray II and Apollo II (for electrospray ionization in positive and negative modes).
• Q‑region omni‑directional accumulation (Q2, Q5) enabling resonance excitation (RCID), collision‑based fragmentation (CCID, xCID) and MSn isolation prior to ToF detection.
• OmniScape (OSc) software for systematic linearization, PTM/termini variant screening and scoring/annotation of fragment spectra.

Methods

• Ionization and mobility separation: Cyclic peptides were ionized in positive or negative electrospray mode. After source activation, ion mobility separation in the TIMS cartridge filters species by collisional cross section (CCS) before mass selection.
• Fragmentation in Omnitrap: Selected precursors were accumulated in omnidirectional traps and fragmented using multiple activation methods—collision‑induced dissociation variants (xCID, CCID, RCID) and electron‑based activation using the Omnitrap electron source. This approach promotes ring opening and production of sequence‑informative linear fragments (b/y, a/x, c/z types).
• Systematic computational linearization: Because a cyclic peptide can be linearized at any peptide bond (producing multiple distinct N/C‑terminal configurations) and can undergo termini modifications (e.g., water elimination, diverse terminal chemistries), OSc generates all feasible linearized sequence representations (for an 11‑residue macrocycle this implies dozens of candidate linear sequences and termini variants). Each candidate is used to generate theoretical fragment libraries.
• Scoring and confirmation: Experimental MS2 spectra are exhaustively compared to all candidate linearizations and termini/PTM variants. Multiple independent scoring metrics and fragment annotations are used to rank candidates and confirm the top‑scoring linearized sequence. Low‑abundance fragments matching alternative linearizations are retained and can be quantified to evaluate fragmentation branching ratios.

Main results and discussion

• Exhaustive coverage and confident assignment: For cyclosporin A (an 11‑residue cyclic peptide family), the OSc workflow identified a single top‑scoring linearized sequence (CSA6) with 100% sequence coverage except for a single missing y‑type cleavage. Alternative high‑scoring linearizations (e.g., CSA5, CSA8) were detected and characterized, with CSA5 lacking several b‑type fragments despite 100% coverage otherwise.
• Comprehensive fragment explanation: The combination of TIMS filtering, targeted accumulation and multiple fragmentation flavors produced dense MS2 spectra containing b/y, a/x and c/z fragments. OSc matched and annotated major and lower‑abundance peaks, enabling a full‑spectrum explanation and localization of preferential cleavage sites.
• Quantification of fragmentation pathways: Lower‑abundance fragments attributable to alternative ring‑opening sites were assigned, demonstrating the ability to quantify the relative contributions (branching ratios) of competing fragmentation routes within a single MS2 spectrum.
• Generality and transferability: The timsOmni–OSc approach was successfully applied to additional cyclic peptide derivatives, supporting its broader applicability to macrocycles that contain non‑canonical residues or that resist digestion with proteases.

Benefits and practical applications

• High confidence sequence assignment for cyclic peptides that are not amenable to proteolytic digestion or classical bottom‑up workflows.
• Ability to identify and localize PTMs and termini chemistries arising from cyclization (e.g., water elimination variants), which is crucial for synthetic products and impurity profiling.
• Detailed mapping of fragmentation preferences and minor pathways, enabling mechanistic insight and potential quantification of isomeric or isobaric species.
• Relevance to drug discovery and quality control: supports lead identification, structure confirmation, batch release testing and impurity characterization of cyclic peptide therapeutics and related macrocycles.

Future trends and potential applications

• Integration with machine learning: applying ML models to prioritize candidate linearizations and refine scoring will reduce computational load and improve throughput for large libraries.
• Automation and high‑throughput pipelines: coupling TIMS‑Omnitrap workflows with automated sample handling and OSc batch processing will facilitate screening of large synthetic libraries.
• Extension to larger and more complex macrocycles: adapting activation regimes and software rules to handle crosslinks, stapled peptides and non‑peptidic macrocycles.
• Quantitative branching analysis: development of standardized approaches to quantify relative contributions of alternative fragmentations and to use these metrics in SAR studies.
• Regulatory and QC adoption: with further validation, comprehensive top‑down cyclic peptide characterization could become part of regulatory dossiers for peptide therapeutics.

Conclusion

The timsOmni platform combined with OmniScape processing provides a robust, general and high‑confidence top‑down workflow for cyclic peptide sequencing. By combining ion mobility separation, multiple targeted fragmentation strategies in the Omnitrap and exhaustive computational linearization with PTM/termini screening, the method yields near‑complete sequence coverage, precise fragment annotation and the ability to detect and quantify alternative ring‑opening pathways. This approach is well suited to the analytical challenges posed by cyclic peptide discovery and development.

References

• Bartelmus C., Greisch J.‑F., Smyrnakis A., Kosmopoulou M., Suckau D., Tremintin G., Papanastasiou D., Greig M. Comprehensive sequencing of cyclic peptides by top‑down MS on a timsOmni platform using next‑generation fragmentation and OmniScape processing. ASMS 2026 (conference presentation/data summary). For Research Use Only. Not for use in diagnostic procedures.