Fluorescent Glycan Fingerprinting of SARS2 Spike Proteins

  1. Zhengliang L Wu
  2. James M Ertelt

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Abstract

Glycosylation is the most common post-translational modification and has myriad biological functions. However, glycan analysis and research has always been a challenge. Here, we would like to present new techniques of glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS-2 spike (S) glycoproteins. SARS-2, a novel coronavirus and the causative agent of COVID-19 pandemic, has devastated the world since the end of 2019. To obtain the N-glycan fingerprint of a S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, and then separated on acrylamide gel through electrophoresis, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders that are enzymatically generated are run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. Due to lack of enzyme for broad O-glycans releasing, O-glycans on the RBD protein are labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated on gel. Glycan fingerprinting could serve as a quick way for global assessment of the glycosylation of a glycoprotein.

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    SciScore for 10.1101/2021.01.16.426965: (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.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Different recombinant SARS-CoV-2 Spike RBD proteins expressed in HEK293 cells, Tn5 insect cells, CHO cells, full length recombinant SARS-CoV-2 Spike proteins expressed in HEK293 cells and CHO cells, and recombinant SARS-CoV-2 Spike S1 subunit protein expressed in HEK293 cells were from Bio-Techne.
    HEK293
    suggested: None
    CHO
    suggested: None

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.01.16.426965v1 on bioRxiv