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

Lucia Grenga; Fabrice Gallais; Olivier Pible; Jean-Charles Gaillard; Duarte Gouveia; Hélène Batina; Niza Bazaline; Sylvie Ruat; Karen Culotta; Guylaine Miotello; Stéphanie Debroas; Marie-Anne Roncato; Gérard Steinmetz; Charlotte Foissard; Anne Desplan; Béatrice Alpha-Bazin; Christine Almunia; Fabienne Gas; Laurent Bellanger; Jean Armengaud

The MS/MS spectra recorded on each sample were assigned to peptide sequences using the Mascot Server 2.5.1 (Matrix Science). A database (20,585 sequences; 10,847,418 residues) containing the UniProt Chlorocebus sequences (downloaded March 2020) and the Italy-INMI1 SARS-CoV-2 protein sequences was queried after a first analysis to remove contaminant spectra against an in-house 'common contaminants' database (384 sequences; 187,250 residues) encompassing 361 contaminants classically observed in proteomics (cRAP + additional contaminants) and 23 Bos taurus sequences corresponding to the most abundant proteins from foetal calf serum present in the cell culture medium [16] . Peptide-to-MS/MS spectrum assignation was done with the following parameters: full trypsin specificity, maximum of two missed cleavages, mass tolerances on the parent ion of 5 ppm and 0.02 higher than 98% of the top ion score. Proteins were grouped if they shared at least one peptide, and in each group label-free quantification was based on PSM counts for each protein following the principle of parsimony. Proteins identified by one or more specific peptides were retained for the analysis (protein FDR 1%).