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Authors: Zhou D et al. Link to paper:

Journal/ Pre-Print: Preprints

Key Words: N-glycosylation, spike protein, glycopeptide, mass spectrometry, epitope prediction


1. 22 N-glycosylation sites were found in the recombinant Spike protein of SARS-CoV-2 secreted from BTI-Tn-5B1-4 cells

2. 8 N-glycosylation sites are located in the N-terminal domain (NTD), 2 in the receptor binding domain (RBD), 3 in the rest of S1 subunit and 9 are located in the S2 subunit

3. “Achilles heel” – glycan free density, YQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQ, was identified in RBD of SARS-CoV- 2 in the interface to ACE2


This paper identifies N-glycosylation sites of the recombinant SARS-CoV-2 Spike protein expressed by insect BTI-Tn-5B1-4 cells. It identifies the same 22 N-glycosylation sites, which are in agreement with previous published papers: (; 


Develop a vaccine for SARS-CoV2/COVID19


· In silico study / bioinformatics study

· In vitro study


Novelty: No novelty

Standing in the field: 2 previous publications show similar results.

Appropriate statistics: Yes

Viral model used: Expression of spike protein of SARS-CoV-2 in insect BTI-Tn-5B1-4 cells.

Translatability: Potential epitopes for neutralising antibodies.

Main limitations: This type of work has been published by other groups to greater level of sophistication and detail. These have been carried out protein expression in human cells and they are more confident that the recombinantly produced protein is in the trimer form, as well as backing up the MS data with UPLC glycan profiling. There is agreement that all 22 potential sites are occupied and mainly polymannose (which are unsurprisingly observed in this publication as insect cells are used as the expression system). Ideally this would be carried out with proteins from a purified virus as this would allow the native glycosylation to be observed, as the virus may well assemble and bud quite differently to an overexpressed secreted protein. However, this is of course more challenging.