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.
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