UTILISATION OF CYCLIC ION MOBILITY WITH MULTIPLE PASS ACQUISITION FOR THE ANALYSIS OF GLYCOPEPTIDES AND GLYCOFORMS ASSOCIATED WITH SARS-COV-2

Significance of the Topic

Glycosylation of the SARS-CoV-2 spike protein plays a critical role in immune evasion and receptor binding. Detailed characterization of glycopeptides and glycoforms supports vaccine design and therapeutic monitoring. High-resolution ion mobility separation offers enhanced distinguishing of co-eluting glycoforms, improving glycoproteomic depth.

Objectives and Study Overview

This work aims to evaluate cyclic ion mobility spectrometry (cIMS) with multi-pass acquisition for in-depth analysis of SARS-CoV-2 spike glycopeptides. The study compares single-pass and multi-pass modes to resolve isobaric glycoforms and achieve high sequence coverage.

Methodology and Instrumentation

• Sample Preparation: Tryptic digestion of recombinant SARS-CoV-2 spike protein.
• Chromatography: NanoLC on 75 µm column with 45-minute gradient (0.1% FA in water to 0.1% FA in ACN).
• Mass Spectrometry: Waters Cyclic IMS coupled to TOF-MS in HDMSE mode (19–45 eV collision energy).
• Data Analysis: ProteinLynx Global Server for peptide identification and glycoform assignment.

Main Findings and Discussion

• Single-pass HDMSE provided 75% sequence coverage of the spike protein and allowed initial glycopeptide profiling.
• Multi-pass cIMS resolved co-eluting glycoforms by separating species with identical m/z but different drift times.
• High-resolution IMS distinguished sialic acid linkage variants on glycopeptides, revealing positional isomers.
• Mirror plots of MS/MS spectra showed identical fragmentation for isobaric glycoforms, highlighting the necessity of ion mobility for differentiation.

Benefits and Practical Applications

• Enhanced glycoproteomic characterization for viral antigen analysis.
• Improved detection of glycan heterogeneity critical for vaccine antigen quality control.
• Flexible acquisition workflow enabling rapid profiling followed by targeted structural elucidation.

Future Trends and Potential Applications

• Integration of multi-pass ion mobility with targeted glycan quantitation workflows.
• Expansion to other viral glycoproteins and complex biotherapeutic glycosylation studies.
• Development of advanced data analysis tools for automated glycoform assignment using mobility dimensions.

Conclusion

Cyclic IMS with multi-pass acquisition significantly improves the resolution of glycopeptide isomers in SARS-CoV-2 spike analysis. The approach yields high sequence coverage and unravels glycoform complexity, supporting vaccine development and glycoproteomic research.

References

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