Site-Specific Characterization of the O-linked Glycans at the Furin Cleavage Site of the Sars-CoV-2 Spike Protein using Cyclic IMS

Importance of the Topic

The O-glycosylation pattern proximal to the furin cleavage site of the SARS-CoV-2 spike protein is a critical determinant of viral activation and cell entry. Site-specific structural insights into these glycans can inform vaccine development, antiviral strategies, and diagnostic assay design.

Objectives and Overview

This study aims to characterize four distinct O-glycoforms attached to threonine 56 in the S1/S2 cleavage region of the SARS-CoV-2 spike protein. Using cyclic ion mobility spectrometry coupled with targeted MS/MS, the work delineates isomeric glycan structures and linkage variants.

Methods and Instrumentation

Peptide Preparation:

• Reduction with 5 mM DTT and alkylation with 15 mM iodoacetamide, followed by digestion with trypsin and PNGase F.
• NanoLC separation on a Waters nanoEase HSS T3 100 Å, 1.8 µm, 75 µm×15 cm column with a Symmetry C18 trap (5 µm, 100 Å).
• Mobile phases: 0.1% formic acid with 1 ppm citric acid in water (A) and acetonitrile (B); gradient from 10% to 35% B over 60 min.

Targeted MS Workflow:

• Quadrupole isolation and collisional activation in the trap region.
• Ion mobility separation in a Waters Cyclic IMS cell using one and five passes optimized for trisaccharide fragments.

Instrumentation

• Waters Cyclic IMS system for high-resolution ion mobility separation.
• Waters nanoACQUITY UPLC for nanoLC peptide separation.

Main Results and Discussion

• MS/MS spectra of four glycopeptides revealed oxonium ions at m/z 528, 657, 819, and 1022.
• Single-pass IMS distinguished core 1 and core 2 fragments; five-pass IMS resolved NeuAcα2-6Galβ1-4GlcNAc (arrival time ~73.2 ms) from NeuAcα2-3Galβ1-3GalNAc (~73.8 ms) and a second α2-3Galβ1-4GlcNAc isomer (~80.2 ms).
• Penta- and hexasaccharide glycoforms exhibited similar mobility distributions, suggesting conserved conformation across extended core 1 structures.
• Findings confirm the site-specific presence of core 1, core 2, and extended core 1 O-glycan structures at T56.

Benefits and Practical Applications

This approach provides unambiguous structural assignment of isomeric O-glycans adjacent to the furin cleavage site, advancing our understanding of viral glycosylation. It is directly applicable to vaccine design, therapeutic antibody characterization, and quality control in biopharmaceutical production.

Future Trends and Opportunities

• Integration with exoglycosidase digestion and orthogonal fragmentation methods for deeper glycan profiling.
• Application of multi-pass IMS to other viral glycoproteins and complex biological samples.
• Automation and high-throughput workflows for clinical glycomics and biopharmaceutical analysis.

Conclusion

Cyclic IMS combined with targeted MS/MS enables detailed, site-specific characterization of O-glycan isomers at the SARS-CoV-2 furin cleavage site. The method resolves linkage-specific isomers and confirms the presence of diverse core structures, offering a powerful tool for glycoprotein research.

References

• Hoffmann M, Kleine-Weber H, Pöhlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Molecular Cell. 2020;78(4):779–784.
• Guttman M, Lee KK. Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry. Analytical Chemistry. 2016;88(10):5212–5217.