Comprehending COVID-19: Structural Characterization of the Glycan Assemblies of O-linked Glycopeptides Using Cyclic Ion Mobility

Importance of the topic

The glycosylation of viral envelope proteins plays a critical role in infectivity, immune evasion, and host recognition. O-linked glycans near the SARS-CoV-2 spike protein furin cleavage site are particularly challenging to characterize due to low abundance, structural heterogeneity, and lack of consensus motifs. Detailed linkage and site-specific information can guide vaccine design and therapeutic strategies against COVID-19.

Study objectives and overview

This work aims to exploit the Waters SELECT SERIES Cyclic IMS instrument to achieve site- and linkage-specific structural characterization of low-level O-linked glycopeptides derived from the SARS-CoV-2 spike protein. The focus is on glycopeptides spanning the furin cleavage region to understand core 1, extended core 1, and core 2 glycan assemblies and their sialic acid linkages.

Methodology

Recombinant SARS-CoV-2 spike protein expressed in HEK 293 cells was reduced, alkylated, deglycosylated (PNGaseF), and digested with trypsin. Nano-LC separation was performed on a UPLC M-Class system with reversed-phase columns maintained at 60 °C, using a water–acetonitrile gradient with formic and citric acids. Mass spectrometry analysis employed ESI+ on the SELECT SERIES Cyclic IMS with targeted MS/MS fragmentation in the trap region. Oxonium ions were separated by cyclic ion mobility with up to five passes to resolve isomeric structures. Data were processed using DriftScope and MassLynx software platforms.

Used instrumentation

• ACQUITY UPLC M-Class system
• SELECT SERIES Cyclic IMS mass spectrometer
• nanoEase M/Z HSS T3 and Symmetry C18 columns
• MassLynx, DriftScope, and Embedded Analyzer software

Main results and discussion

MS/MS fragmentation of the T56 glycopeptide revealed distinct oxonium ion profiles for glycoforms A–D. Single-pass ion mobility separated two mobility populations corresponding to α2-3 and α2-6 sialylated HexNAc-Hex-NeuAc isomers. Five-pass separations further resolved substructures, confirming extended core 1 and core 2 glycan motifs. Collision cross sections matched literature values for NeuAcα2-3Galβ1-4GlcNAc (247.2 Å2) and NeuAcα2-6Galβ1-4GlcNAc (236.9 Å2). Chromatographic heterogeneity indicated multiple isomeric glycopeptides eluted from reversed-phase columns, highlighting glycan microheterogeneity at the furin site.

Benefits and practical applications of the method

The combination of targeted CID and scalable cyclic ion mobility delivers site-specific and linkage-specific glycan structural information at low abundance. This enhances profiling of viral glycoproteins, accelerates vaccine and immunotherapy research, and offers a robust tool for industrial QA/QC of glycoprotein therapeutics.

Future trends and applications

Emerging trends include integrating ion mobility with electron-based fragmentation to map glycopeptide branching, high-throughput workflows for comprehensive glycoproteome analysis, and coupling with bioinformatics for automated glycan annotation. The approach can be extended to other viral and human glycoproteins to elucidate glycan function in disease and therapeutic development.

Conclusion

The SELECT SERIES Cyclic IMS instrument enables unprecedented structural resolution of O-linked glycopeptides from the SARS-CoV-2 spike protein. Scalable ion mobility and targeted fragmentation provide detailed insights into glycan linkages and sites, supporting efforts to combat COVID-19 through vaccine and drug design.

References

1. Johns Hopkins Coronavirus Resource Center. COVID-19 global case tracker.
2. Andersen KG et al. The proximal origin of SARS-CoV-2. Nat Med. 2020;26:450–452.
3. Tortorici AM, Veesler D. Structural insights into coronavirus entry. Adv Virus Res. 2019;105:93–116.
4. Walls AC et al. Structure, function, and antigenicity of SARS-CoV-2 spike glycoprotein. Cell. 2020;181:281–292.e6.
5. Clark GF. The role of glycans in immune evasion. Mol Hum Reprod. 2014;20:185–199.
6. Chang D, Zaia J. Why glycosylation matters in building a better flu vaccine. Mol Cell Proteomics. 2019;18:2348–2358.
7. Khatri K et al. Influenza A virus glycan microheterogeneity and host interactions. Mol Cell Proteomics. 2016;15:1895–1912.
8. Kumar S et al. Structural, glycosylation and antigenic variation between 2019-nCoV and SARS-CoV. Virusdisease. 2020;31:13–21.
9. Watanabe Y et al. Site-specific glycan analysis of the SARS-CoV-2 spike. Science. 2020;eabb9983.
10. Shajahan A et al. Deducing the N- and O-glycosylation profile of SARS-CoV-2 spike protein. Glycobiology. 2020.
11. Sanda M, Morrison L, Goldman R. N- and O-glycosylation of the SARS-CoV-2 spike protein. bioRxiv. 2020;2020.07.05.187344.
12. Zhang Y et al. Site-specific N-glycosylation characterization of recombinant SARS-CoV-2 spike proteins. Mol Cell Proteomics. 2020.
13. Zhou D et al. Identification of 22 N-glycosites on SARS-CoV-2 spike glycoprotein. Glycobiology. 2020;cwaa052.
14. Hoffmann M et al. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection. Mol Cell. 2020;78:779–784.
15. Xia S et al. The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion. Signal Transduct Target Ther. 2020;5:92.
16. Guttman M, Lee KK. Site-specific mapping of sialic acid linkage isomers by IMS. Anal Chem. 2016;88:5212–5217.
17. Depland AD et al. Identification of sialic acid linkage isomers using IRMPD and MS. Int J Mass Spectrom. 2018;434:64–69.