Recombinant SARS-CoV-2 Receptor Binding Domain: Comprehensive Top-Down Sequence Confirmation, Curation and O-Glycosylation Site Determination

Importance of the Topic

The receptor binding domain (RBD) of the SARS-CoV-2 spike protein is central to viral entry via ACE2 interaction and represents a key antigen for diagnostics, vaccine design and therapeutic research. Post-translational modifications, in particular N- and O-linked glycosylation, influence binding affinity, antigenicity and immune evasion. Accurate characterization of recombinant RBD preparations is therefore essential to ensure consistency and reliability of downstream applications.

Objectives and Study Overview

This study aimed to perform an in-depth top-down mass spectrometric analysis of two commercially produced recombinant SARS-CoV-2 RBDs derived from Chinese hamster ovary (CHO) and human embryonic kidney (HEK293) cells. The goals were:

• To confirm and curate the primary amino acid sequences of each RBD construct.
• To localize O-glycosylation sites with single‐residue resolution.
• To provide validated sequences for subsequent glycan profiling and functional assays.

Methodology and Instrumentation

RBD samples bearing a C-terminal His6 tag were enzymatically deglycosylated (PNGase F for N-glycans; endo-α-N-acetylgalactosaminidase OglyZOR and sialidase SialEXO for O-glycans), reduced with DTT and submitted to intact mass analysis. Top-down sequence confirmation employed MALDI in-source decay (MALDI-ISD) on two platforms:

• rapifleX MALDI-TOF/TOF (Bruker) using sDHB matrix and Compass for flexSeries for data processing.
• timsTOF fleX (Bruker) with ion mobility QTOF for high‐resolution low-mass fragment detection.

Spectra were analyzed using flexAnalysis, BioPharma Compass and Biotools software to annotate c- and y-ion series and identify sequence variants and glycoforms.

Main Results and Discussion

CHO-expressed RBD matched the theoretical mass (25 922.96 Da) and yielded 62 % sequence coverage by MALDI-ISD, confirming its amino acid sequence.
HEK293-expressed RBD exhibited an unexpected +110.8 Da mass shift at the N-terminus. Automated clipping analysis of the pro-peptide region revealed an extra pyro-glutamate–modified residue (pQ) following signal peptide removal. Incorporation of this modification restored sequence agreement.
O-glycosylation analysis identified a single site at Thr-6 (corresponding to Thr-323 in full‐length spike numbering) carrying core 1 and core 2 structures. The timsTOF fleX platform successfully resolved c-ion fragments below m/z 1000, enabling unambiguous assignment of Thr-6 glycosylation and excluding Ser-8.

Benefits and Practical Applications

• Validated RBD sequences with defined glycosylation support reproducible production of antigens for serological assays and vaccine studies.
• Top-down MS workflows accelerate detection of unexpected sequence variants and modifications that may alter antigen performance.
• High-resolution glycosite mapping informs design of glycoengineered antigens with optimized immunogenicity.

Future Trends and Potential Uses

Integration of ion mobility‐enhanced Top-Down sequencing will expand capabilities for full proteoform profiling of viral and biotherapeutic proteins. Automated real‐time quality control using high‐throughput MALDI-ISD could be applied to manufacturing workflows. Combining Top-Down data with advanced glycoproteomics and native MS will provide holistic views of antigen heterogeneity and functional impacts.

Conclusion

Top-down MALDI-ISD sequencing on rapifleX and timsTOF fleX platforms enabled comprehensive sequence confirmation and O-glycosylation site localization of recombinant SARS-CoV-2 RBDs from CHO and HEK293 cells. The curated pQ-HEK293-RBD sequence and Thr-6 glycosylation assignment lay the groundwork for detailed N- and O-glycan profiling and ensure reliable antigen quality for research and diagnostic use.

Used Instrumentation

• rapifleX MALDI-TOF/TOF mass spectrometer (Bruker Daltonics)
• timsTOF fleX ion mobility QTOF (Bruker Daltonics)
• flexAnalysis (Compass for flexSeries)
• BioPharma Compass 2021 (Bruker Daltonics)
• Biotools 3.2 SR7 (Bruker Daltonics)

References

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