Computational protocol: Emmental Cheese Environment Enhances Propionibacterium freudenreichii Stress Tolerance

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Protocol publication

[…] Analyses of variance (ANOVA) were performed with the significance level set at P≤0.05, either using growth medium as one factor with FactoMineR, an R package [], or two factors using medium culture and stage of growth. Newman Keuls test was performed as post ANOVA analysis. [...] Gel pieces were washed with acetonitrile and ammonium bicarbonate solution, and then dried under vacuum in a SpeedVac concentrator (SVC100H-200; Savant, Thermo Fisher Scientific, Waltham, MA, USA). In-gel trypsin digestion was performed overnight at 37°C and stopped with spectrophotometric-grade trifluoroacetic acid (TFA) (Sigma-Aldrich) as described previously []. The supernatants containing peptides were then vacuum dried in Speed-Vac concentrator and stored at -20°C until mass spectrometry analysis.Nano-LC-MS/MS analysis was performed using an on-line liquid chromatography tandem mass spectrometry (MS/MS) setup using a Dionex U3000-RSLC nano-LC system fitted to a QSTAR XL (MDS SCIEX, Ontario, Canada) equipped with a nano-electrospray ion source (ESI) (Proxeon Biosystems A/S, Odense, Denmark). Samples were first concentrated on a PepMap 100 reverse-phase column (C18, 5 μm particle size, 300-μm inner diameter (i.d.) by 5 mm length) (Dionex, Amsterdam, The Netherlands). Peptides were separated on a reverse-phase PepMap 100 column (C18, 3 μm particle size, 75 μm i.d. by 150 mm length) (Dionex) at 35°C, using solvent A (2% (vol/vol) acetonitrile, 0.08% (vol/vol) formic acid, and 0.01% (vol/vol) TFA in deionized water) and solvent B (95% (vol/vol) acetonitrile, 0.08% (vol/vol) formic acid, and 0.01% (vol/vol) TFA in deionized water). A linear gradient from 10 to 40% of solvent B in 17 min was applied for the elution at a flow rate of 0.3 μL min-1. Eluted peptides were directly electrosprayed into the mass spectrometer operated in positive mode. A full continuous MS scan was carried out followed by three data-dependent MS/MS scans. Spectra were collected in the selected mass range 300 to 2 000 m/z for MS and 60 to 2 000 m/z for MS/MS spectra. 2+ to 4+ charged ions were considered for the MS/MS analysis when ion intensity was above 10 cps. The mass spectrometer was operated in data-dependent mode automatically switching between MS and MS/MS acquisition using Analyst QS 1.1 software. The instrument was calibrated by multipoint calibration using fragment ions that resulted from the collision-induced decomposition of a peptide from β-casein, β-CN (193–209).To identify peptides, all data (MS and MS/MS) were submitted to X! Tandem using the X! Tandem pipeline developed by PAPPSO (Plateforme d'Analyse Protéomique de Paris Sud-Ouest (PAPPSO), INRA, Jouy-en-Josas, France, search was performed against a database composed of (i) a homemade database containing all the predicted proteins of the P. freudenreichii strain CIRM-BIA 1 used in this study and (ii) a portion of the UniProtKB database corresponding to P. freudenreichii.Database search parameters were specified as follow: trypsin cleavage was used and the peptide mass tolerance was set to 0.2Da for both MS and MS/MS. Oxidation of methionine and deamidation of asparagine and glutamine residues were included as variable modifications. Protein identifications were automatically validated when they showed at least two unique peptides with an e-value below 0.05. […]

Pipeline specifications

Software tools FactoMineR, X! Tandem
Databases UniProtKB
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Propionibacterium freudenreichii
Chemicals Aspartic Acid, Pyruvic Acid