Confident Characterization of N- and O-Linked Glycopeptides Using Electron-Based Dissociation on a timsOmni Platform

Significance of the topic

Glycoproteomics requires reliable methods that preserve both peptide backbone information and glycan structural detail. Accurate localization of glycosylation sites (especially O-linked) and characterization of glycan composition/structure (critical for N-glycans) underpin biomarker discovery, biotherapeutic quality control, and basic research into protein function. The study evaluates trapped electron-driven dissociation (tExD) workflows on the timsOmni platform to deliver tunable fragmentation that balances glycan preservation with extensive backbone cleavage, addressing a key analytical challenge in glycopeptide MS/MS.

Aims and overview of the study

The work aimed to assess the performance of multiple electron-based and hybrid fragmentation modes implemented in the Omnitrap/timsOmni architecture for confident characterization of N- and O-linked glycopeptides. Specific goals included: optimizing fragmentation by precursor charge state and assisted collision energy; comparing ECciD and EIciD performance for identifications and structural detail; and demonstrating workflows that enable both site localization and glycan structural elucidation in targeted DDA-PASEF experiments.

Methodology

Key experimental design elements:

• Samples: synthetic N- and O-linked glycopeptide standards and glycoprotein digests.
• Acquisition: DDA-PASEF mode with targeted inclusion lists using the timsOmni platform. Ion accumulation and mobility separation were performed by TIMS.
• Fragmentation: multiple electron-driven and hybrid modes were evaluated — ECD, hECD, EID, ECID, EIciD, ECciD and EXciD/hybrid variants — with tunable electron energy (~2–35 eV) and supplemental collision energies adjusted per precursor charge state.
• Charge-state considerations: precursors of varying charge states were tested; fragmentation efficiency of radical species required charge-state-dependent assisted collision energies.
• Data processing: primary automated processing with FragPipe (MSFragger-Glyco, MS-Shepherd, O-Pair), complementary validation with Byonic, and manual spectral annotation in OmniScape, Glypy, and GlycoWorkbench.

Used Instrumentation

Instrumentation and software components reported:

• timsOmni platform incorporating TIMS for ion mobility and Omnitrap for selectable electron-based dissociation.
• nanoElute LC for nano-flow peptide separation prior to ion mobility.
• Electron-based dissociation modes: ECD, hECD, EID, ECID, EIciD, ECciD, EXciD (hybrid).
• Acquisition mode: DDA-PASEF with targeted inclusion lists.
• Data analysis software: FragPipe (MSFragger-Glyco, MS-Shepherd, O-Pair), Byonic, OmniScape, Glypy, GlycoWorkbench.

Main results and discussion

Fragmentation behavior and analytical outcomes summarized from the study:

• Glycan preservation vs backbone fragmentation: tExD modes, particularly ECciD, preserved glycan attachments on peptide backbone fragment ions (c and z• series), enabling confident site localization for O-linked glycopeptides. By contrast, CID-like fragmentation primarily produced glycan loss, hindering localization.
• N-linked glycopeptides: ECciD and EIciD produced rich glycan fragmentation useful for structural analysis. ECciD generated more high-mass Y ions (glycan-bearing fragments), improving characterization of glycan composition and connectivity.
• Backbone coverage: ECciD offered extensive backbone cleavage resulting in high sequence coverage, which supports both site localization and peptide identification.
• Charge-state and energy tuning: Efficient generation of radical-driven fragments depended strongly on precursor charge and on tuned assisted collision energy; the platform permits this optimization to balance radical and collisional pathways.
• Comparative identification performance: Targeted DDA-PASEF experiments showed that ECciD and EIciD achieved comparable numbers of glycoPSMs, glycopeptide precursors, and unique glycopeptide identifications. However, ECciD provided enhanced structural detail (more high-mass Y ions and improved backbone fragmentation).
• Practical spectra handling: Automated search pipelines (MSFragger-Glyco/O-Pair) combined with manual validation and annotation enabled reliable assignment of glycopeptide spectra produced by electron-driven modes.

Benefits and practical applications of the method

Practical advantages demonstrated by the tExD implementation on timsOmni:

• Confident O-glycosite localization: preservation of glycan-bearing backbone fragments enables unambiguous mapping of O-glycosylation, a historically difficult problem.
• Detailed N-glycan structural information: high-mass Y ions and complementary glycan fragmentation facilitate glycan composition and connectivity assignments for N-linked species.
• Flexible workflows: charge-state-dependent tuning and selectable electron modes support targeted, high-throughput analyses across glycopeptide classes.
• Compatibility with existing pipelines: integration with FragPipe and Byonic allows automated identification, while manual tools support expert validation.
• Applications: glycoproteomics research, biopharmaceutical quality control (glycoform monitoring), biomarker discovery, and detailed characterization of recombinant glycoproteins.

Future trends and potential applications

Anticipated developments and opportunities arising from this approach:

• Automation of tuning: implementation of intelligent, charge-state-aware methods that automatically set assisted collision energies and electron parameters for optimal fragmentation.
• Deeper integration with multi-omics: combining tExD glycoproteomics with proteomics and glycomics workflows to deliver richer biological context.
• Improved spectral libraries and AI: expansion of glycopeptide spectral libraries and application of machine learning for rapid annotation of electron-driven glycopeptide spectra.
• Higher throughput clinical translation: adaptation of targeted tExD workflows for routine QC of therapeutic glycoproteins and clinical biomarker assays.
• Enhanced hybrid fragmentation strategies: further development of EXciD-style hybrids to maximize simultaneous glycan and peptide structural information.

Conclusion

The timsOmni implementation of trapped electron-driven dissociation (tExD), and specifically ECciD, provides a tunable, high-performance solution for simultaneous preservation of glycan moieties and extensive peptide backbone fragmentation. This balance enables confident localization of O-linked sites and detailed structural elucidation of N-linked glycans while maintaining comparable identification rates to other hybrid approaches. Charge-state-dependent tuning and compatibility with automated and manual analysis tools make the approach adaptable for research and applied glycoproteomics workflows.

References

• Bai H., Barla I., Grigoriadis A., et al., Confident Characterization of N- and O-Linked Glycopeptides Using Electron-Based Dissociation on a timsOmni Platform (Bruker application note WP396), 2026.