Shotgun proteomics of SARS-CoV-2 infected cells and its application to the optimisation of whole viral particle antigen production for vaccines

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1. 

   ScreenIT

   Jun 5, 2020

   SciScore for 10.1101/2020.04.17.046193: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and Virus preparation: Vero E6 (ATCC, CLR-1586) cells were cultured at 37°C in 9% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco™, ThemoFisher) supplemented with 5% fetal calf serum (FCS) and 0.5% penicillin–streptomycin.	Vero E6 suggested: None
   Infection: For the kinetic, 1×106 Vero cells seeded into 25 cm2 flasks were grown to cell confluence in 5 mL DMEM supplemented with 5% FCS and 0.5% penicillin–streptomycin …

   SciScore for 10.1101/2020.04.17.046193: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture and Virus preparation: Vero E6 (ATCC, CLR-1586) cells were cultured at 37°C in 9% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco™, ThemoFisher) supplemented with 5% fetal calf serum (FCS) and 0.5% penicillin–streptomycin.	Vero E6 suggested: None
   Infection: For the kinetic, 1×106 Vero cells seeded into 25 cm2 flasks were grown to cell confluence in 5 mL DMEM supplemented with 5% FCS and 0.5% penicillin–streptomycin for one night at 37°C under 9% CO2.	Vero suggested: None
   Software and Algorithms
   Sentences	Resources
   Secondary ions were isolated with a window of 2.0 m/z. MS/MS Data Interpretation and label free protein quantification: The MS/MS spectra recorded on each sample were assigned to peptide sequences using the Mascot Server 2.5.1 (Matrix Science).	Mascot suggested: (Mascot, RRID:SCR_014322)
   Mascot DAT files were parsed using the Python version of Matrix Science msparser version 2.5.1 with function ms_peptidesummary.	Python suggested: (IPython, RRID:SCR_001658)
   MS/MS data repository: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [17] partner repository with the dataset identifier PXD018594 and 10.6019/PXD018594.	PRIDE suggested: (Pride-asap, RRID:SCR_012052)
   Co-expression cluster analysis was obtained using the Bioconductor R package coseq v1.5.2 [19].	Bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   Statistically enriched (FDR ≤ 0.05) GO terms on proteins that are differentially expressed between pairwise samples or on proteins assigned to each co-expression cluster were identified using Metascape [20].	Metascape suggested: (Metascape, RRID:SCR_016620)
   The most statistically enriched GO terms were visualized in ggplot2 [21].	ggplot2 suggested: (ggplot2, RRID:SCR_014601)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Virus yields from the dedicated cell culture systems could also represent a limitation. Concomitantly with vaccine development, inactivated virus particles are also of interest for testing real serology or screening neutralizing antibodies. Evidently, production of well-characterized active virus particles is also of interest for fundamental research purposes. Given the requirement for speed, here we evaluate the use of LC-MS/MS as a tool for guiding the optimisation of the conditions for SARS-CoV-2 whole viral particle antigen production. The results presented here demonstrate the potential of our pipeline to profile virus production across time. In particular, by analysing the proteome of Vero cells infected with SARS-CoV-2 at two different MOI, it was possible to monitor changes in the levels of three SARS-CoV-2 structural proteins and three non-structural ones. Whilst as for other analyses [11,13] we could not detect peptides from protein E like. The lack of detection of other accessory proteins could be imputed to differences in samples processing with the protocol described here favouring simplified steps and speed while maintaining accuracy. Deeper analyses are envisaged for monitoring virus homogeneity during the different steps of viral production once the most permissive conditions will be established. Remarkably, comparable profiles were obtained at the two tested MOI, with the profiles obtained at lower MOI slightly delayed and hence more insightful regarding the ...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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

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   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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   Read the original source
2. 

   Version published to 10.1101/2020.04.17.046193v1 on bioRxiv

   Apr 17, 2020