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Refining the N-Termini of the SARS-CoV-2 Spike Protein and Its Discrete Receptor-Binding Domain

  1. Author:
    D'Ippolito,Robert [ORCID]
    Drew,Matt
    Mehalko,Jennifer
    Snead,Kelly
    Wall,Vanessa
    Putman,Zoe
    Esposito,Dom
    Dehart,Caroline [ORCID]
  2. Author Address

    NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, United States.,
    1. Year: 2021
    2. Date: Sep 3
    3. Epub Date: 2021 08 11
  1. Journal: Journal of proteome research
    1. 20
    2. Pages: 4427-4434
  2. Type of Article: Article
  3. ISSN: 1535-3893
  1. 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.

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External Sources

  1. DOI: 10.1021/acs.jproteome.1c00349
  2. PMID: 34379411
  3. WOS: 000693638800018

Library Notes

  1. Fiscal Year: FY2020-2021
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