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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Abstract
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and continues to spread quickly around the globe. Currently, no effective vaccine is available to prevent COVID-19 and an intense global development activity is in progress. In this context, the different technology platforms face several challenges resulting from the involvement of a new virus still not fully characterised. Finding of the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles is among them. Here, we describe the establishment of a workflow based on shotgun tandem mass spectrometry data to guide the optimisation of the conditions for viral amplification. In parallel, we analysed the dynamic of the host cell proteome following SARS-CoV-2 infection providing a global overview of biological processes modulated by the virus and that could be further explored to identify drug targets to address the pandemic.
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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 E6suggested: NoneInfection: 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 E6suggested: NoneInfection: 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. Verosuggested: NoneSoftware 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). Mascotsuggested: (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. Pythonsuggested: (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. PRIDEsuggested: (Pride-asap, RRID:SCR_012052)Co-expression cluster analysis was obtained using the Bioconductor R package coseq v1.5.2 [19]. Bioconductorsuggested: (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]. Metascapesuggested: (Metascape, RRID:SCR_016620)The most statistically enriched GO terms were visualized in ggplot2 [21]. ggplot2suggested: (ggplot2, RRID:SCR_014601)Results from OddPub: Thank you for sharing your data.
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 ...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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).
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.
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- No protocol registration statement was detected.
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