Structural O-Glycoform Heterogeneity of the SARS-CoV-2 Spike Protein Receptor-Binding Domain Revealed by Native Top-Down Mass Spectrometry

  1. David S. Roberts
  2. Morgan W. Mann
  3. Jake A. Melby
  4. Eli J. Larson
  5. Yanlong Zhu
  6. Allan R. Brasier
  7. Song Jin
  8. Ying Ge

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) utilizes an extensively glycosylated surface spike (S) protein to mediate host cell entry and the S protein glycosylation is strongly implicated in altering viral binding/function and infectivity. However, the structures and relative abundance of the new O-glycans found on the S protein regional-binding domain (S-RBD) remain cryptic because of the challenges in intact glycoform analysis. Here, we report the complete structural characterization of intact O-glycan proteoforms using native top-down mass spectrometry (MS). By combining trapped ion mobility spectrometry (TIMS), which can separate the protein conformers of S-RBD and analyze their gas phase structural variants, with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analysis, the O-glycoforms of the S-RBD are comprehensively characterized, so that seven O-glycoforms and their relative molecular abundance are structurally elucidated for the first time. These findings demonstrate that native top-down MS can provide a high-resolution proteoform-resolved mapping of diverse O-glycoforms of the S glycoprotein, which lays a strong molecular foundation to uncover the functional roles of their O-glycans. This proteoform-resolved approach can be applied to reveal the structural O-glycoform heterogeneity of emergent SARS-CoV-2 S-RBD variants, as well as other O-glycoproteins in general.

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    SciScore for 10.1101/2021.02.28.433291: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The recombinant SARS-CoV-2 spike regional-binding domain protein expressed in the HEK 293 cell line was purchased from Sino Biological Inc. (cat. 40592-VNAH)
    HEK 293
    suggested: None
    Software and Algorithms
    SentencesResources
    Mass spectra were output from the DataAnalysis 5.3 (Bruker Daltonics) software and analyzed using MASH Explorer.64 Data Analysis: All data were processed and analyzed using Compass DataAnalysis 4.3/5.3 and MASH Explorer.64 Maximum Entropy algorithm (Bruker Daltonics) was used to deconvolute all mass spectra with the resolution set to 80,000 for the timsTOF Pro or with instrument peak width set to 0.05 for the 12T FTICR.
    Compass DataAnalysis
    suggested: None
    For peak picking, the sophisticated numerical annotation procedure (SNAP) algorithm from Bruker DataAnalysis 5.3 was used with a quality threshold of 0.5 and an S/N lower threshold of 3.
    SNAP
    suggested: (SNAP, RRID:SCR_007936)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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.

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  2. Version published to 10.1101/2021.02.28.433291 on bioRxiv