CONFIDENT O-GLYCOSYLATION SITE IDENTIFICATION USING A CYCLIC ION MOBILITY– MASS SPECTROMETER EQUIPPED WITH ECD FUNCTIONALITY

Confident O-Glycosylation Site Identification Using Cyclic IMS–MS with ECD Functionality

Importance of the Topic

O-glycosylation plays a critical role in the structure and function of therapeutic glycoproteins, influencing stability, activity, and immunogenicity. Precise mapping of O-glycan attachment sites is essential for biopharmaceutical characterization, quality control, and regulatory compliance.

Study Objectives and Overview

• Demonstrate the capabilities of electron capture dissociation (ECD) on a cyclic ion mobility spectrometry (IMS)–enabled mass spectrometer for unambiguous assignment of O-glycosylation sites.
• Apply multi-pass IMS and IMSn slicing to resolve and isolate glycopeptide isomers from the biotherapeutic Enbrel (etanercept).
• Highlight the benefits of positioning the ExD cell post-IMS for enhanced gas-phase separation prior to fragmentation.

Methodology and Instrumentation

• Sample Preparation: Tryptic digest of reduced and alkylated Enbrel at 4 µM (infusion) and 2 µM (LC-MS).
• Infusion Experiments: Static nanospray with targeted HDMS/MS ECD; multi-pass IMS followed by IMSn slicing and subsequent ECD fragmentation.
• LC-MS Experiments: ACQUITY I-Class UPLC system, CSH C18 column (2.1 × 100 mm, 1.7 µm) at 60 °C; gradient 1–35% acetonitrile over 25 min; targeted HDMS/ECD acquisition.
• Mass Spectrometer: SELECT SERIES™ Cyclic™ IMS equipped with a post-IMS eMSion ExD cell; enables 1–20 IMS passes to resolve mobility populations.
• Data Analysis: Semi-automated spectrum annotation via ExDViewer BETA v4.2.7.

Results and Discussion

• Infusion Data: Quadrupole isolation of m/z 769 revealed four precursor populations separated by three IMS passes. IMSn slicing of minor component (peak A) with 20 passes provided clear ECD fragment ions that localized the HexNAc–Hex–NeuAc glycan to a single serine residue.
• LC-MS Findings: Extracted ion chromatograms identified two retention peaks at 12.3 min and 12.6 min, corresponding to T200 and S199 glycosylation sites on the tryptic peptide SMAPGAVHLPQPVSTR.
• Glycoform Coverage: Eleven distinct O-glycopeptide structures, including positional isomers, were confidently assigned across the Enbrel digest.
• Instrumental Advantages: Post-IMS ECD enabled preservation of labile glycan–peptide bonds and improved sequence coverage of glycosylated peptides.

Benefits and Practical Applications

• High Confidence in Site Assignment: ECD preserves glycan attachments during backbone cleavage.
• Gas-Phase Separation: Multi-pass IMS and IMSn slicing afford isolation of co-eluting glycoforms without extensive chromatographic methods.
• Biotherapeutic Characterization: Method supports detailed glycoprotein analysis in quality control and R&D settings.

Future Trends and Applications

• Integration with complementary fragmentation techniques (e.g., ETD, CID) for enhanced coverage.
• Advances in IMS resolution and real-time data processing to handle complex glycoproteome samples.
• Automation and informatics platforms for streamlined glycan site mapping workflows.
• Extension to other post-translational modifications and larger glycoprotein systems.

Conclusion

The combination of cyclic IMS, IMSn slicing, and ECD on the SELECT SERIES™ Cyclic™ IMS platform enables robust, gas-phase separation and confident localization of O-glycosylation sites. Applied to Enbrel, this approach successfully resolved positional isomers and mapped eleven unique O-glycoforms, demonstrating its utility for advanced glycoprotein characterization.

Reference

1. Ippoliti S., Cooper-Shepherd D., Yu Y.Q., Langridge J.I. Confident O-glycosylation Site Identification for ENBREL (etanercept) Using the ECD Functionality of SELECT SERIES Cyclic IMS System, Application Note 720007458EN.