Confident O-glycosylation Site Identification for ENBREL (etanercept) Using the ECD Functionality of SELECT SERIES Cyclic IMS System

Significance of the Topic

O-glycosylation is a key post-translational modification affecting stability, activity and immunogenicity of biotherapeutics.
Unlike N-glycosylation, O-glycan attachment sites on serine or threonine residues lack a consensus sequence and are structurally unpredictable.
Accurate mapping of O-glycosylation at the peptide level is crucial for quality control, regulatory compliance and understanding of therapeutic efficacy.

Objectives and Overview of the Study

This work evaluates the Electron Capture Dissociation (ECD) functionality of the SELECT SERIES Cyclic IMS system for unambiguous O-glycosylation site assignment in ENBREL (etanercept).
The study aims to overcome limitations of standard CID fragmentation by retaining labile glycans on peptide backbones and resolving positional isomers.
Eleven distinct O-glycopeptide species were targeted and characterized to demonstrate site-specific mapping capabilities.

Methodology and Instrumentation

A reduced and alkylated tryptic digest of ENBREL was separated by reversed-phase UPLC using an ACQUITY UPLC I-Class System on a CSH C18 column (1.7 µm, 2.1×100 mm) at 60 °C.
Mobile phases comprised 0.1% formic acid in water (A) and acetonitrile (B), with a linear gradient from 1% to 35% B over 25 minutes.
Mass analysis employed a SELECT SERIES Cyclic IMS instrument operated in HDMSE (single pass IMS) and targeted LC-HDMS/MS modes.
ECD fragmentation was performed in a dedicated cell installed post-IMS, generating c- and z-type ions that retain attached glycans for site localization.

Key Results and Discussion

Eleven O-glycopeptide species of ETANERCEPT were detected by MS1 and further probed by ECD MS/MS.
ECD spectra preserved glycan moieties on the peptide backbone and enabled assignment of glycosylation sites via characteristic c- and z-ions.
Example: Isobaric T19 peptide (SMAPGAVHLPQPVSTR + 2 HexNAc + 2 Hex + NeuAc) yielded two chromatographically separated isomers.
Fragment ions c14 and c15 localized sialylated and non-sialylated Core 1 structures to Ser199 and Thr200, respectively, and z-series ions corroborated these assignments.
Additional glycoforms on T1 and T22-23 peptides were similarly mapped, confirming single or multiple O-glycan occupancy per peptide.

Benefits and Practical Applications

ECD provides an orthogonal fragmentation approach to CID, preserving labile O-glycans for direct site mapping.
This method enhances confidence in glycosylation profiling of complex biotherapeutics where multiple, closely spaced sites exist.
Improved sensitivity, resolution and ion mobility separation on the SELECT SERIES Cyclic IMS platform facilitate comprehensive glycopeptide analysis.
Applications extend to quality control, biosimilar characterization and structure–function studies in biopharmaceutical development.

Future Trends and Opportunities

Integration of ECD with high-throughput ion mobility workflows may accelerate site-specific glycoproteomics.
Advanced data analysis algorithms, including machine learning, could automate glycopeptide identification and site assignment.
Expanding ECD applications to other labile modifications and combining with complementary dissociation methods will deepen structural insights.
Broader adoption of cyclic IMS and ECD in regulated laboratories may streamline biotherapeutic characterization and comparability studies.

Conclusion

This study demonstrates that ECD on the SELECT SERIES Cyclic IMS system delivers unambiguous O-glycosylation site assignments in ENBREL.
The approach overcomes CID limitations by retaining glycans on peptide fragments, enabling detailed mapping of 11 O-glycopeptide species.
ECD fragmentation on cyclic IMS emerges as a powerful tool for glycoprotein characterization in research and industrial settings.

References

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