Mass spectrometric based detection of protein nucleotidylation in the RNA polymerase of SARS-CoV-2

  1. Brian J. Conti
  2. Andrew S. Leicht
  3. Robert N. Kirchdoerfer
  4. Michael R. Sussman

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Abstract

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the n on s tructural p rotein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients.

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  1. Version published to 10.1038/s42004-021-00476-4
  2. ScreenIT

    SciScore for 10.1101/2020.10.07.330324: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Gel images were examined using ImageJ opensource software.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    LC-MS peaks were defined by analyzing the data with the feature mapping and precursor quantification nodes in Proteome Discoverer 2.4 that determined the retention time, charge state, and abundance of every peak in each searched file.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    The data were filtered and exported to Microsoft Excel to search for peaks that 1) were not present in unlabeled samples and 2) that possessed two peaks with the same charge state at a similar retention time (+/- 0.5 minute) and were mass-shifted by 4.9852 or 10.0336 amu [MH+ ion masses] for 15N- or 13C-GMP labeling, respectively, within a +/- 1.5 ppm error limit (Extended Data Figure 4, Extended Data Figure 5).
    Microsoft Excel
    suggested: (Microsoft Excel, RRID:SCR_016137)
    Data availability: The raw mass spectrometry datasets and proteome discoverer results that were generated and analyzed in the current study are available through the MassIVE repository under the identifier MSV000085857 [https://doi.org/doi:10.25345/C52B2B].
    MassIVE
    suggested: None

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  3. Version published to 10.1101/2020.10.07.330324v1 on bioRxiv