Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain.
J Proteome Res
; 20(9): 4427-4434, 2021 09 03.
Article
in English
| MEDLINE | ID: covidwho-1351920
ABSTRACT
Previous work employing five SARS-CoV-2 spike protein receptor-binding domain (RBD) constructs, comprising versions originally developed by Mt. Sinai or the Ragon Institute and later optimized in-house, revealed potential heterogeneity which led to questions regarding variable seropositivity assay performance. Each construct was subjected to N-deglycosylation and subsequent intact mass analysis, revealing significant deviations from predicted theoretical mass for all five proteins. Complementary tandem MS/MS analysis revealed the presence of an additional pyroGlu residue on the N-termini of the two Mt. Sinai RBD constructs, as well as on the N-terminus of the full-length spike protein from which they were derived, thus explaining the observed mass shift and definitively establishing the spike protein N-terminal sequence. Moreover, the observed mass additions for the three Ragon Institute RBD constructs were identified as variable N-terminal cleavage points within the signal peptide sequence employed for recombinant expression. To resolve this issue and minimize heterogeneity for further seropositivity assay development, the best-performing RBD construct was further optimized to exhibit complete homogeneity, as determined by both intact mass and tandem MS/MS analysis. This new RBD construct has been validated for seropositivity assay performance, is available to the greater scientific community, and is recommended for use in future assay development.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Spike Glycoprotein, Coronavirus
/
COVID-19
Type of study:
Prognostic study
Limits:
Humans
Language:
English
Journal:
J Proteome Res
Journal subject:
Biochemistry
Year:
2021
Document Type:
Article
Affiliation country:
Acs.jproteome.1c00349
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