Proteinscape 3.1 package

Mass spectrometry (MS) analysis was carried out at the IBMC proteomic platform, Strasbourg. The ribosome sample was analyzed by nano-LC-MS/MS on a NanoLC-2DPlus system (nanoFlexChiP module; Eksigent, ABSciex, Concord, Ontario, Canada) coupled to a TripleTOF 5600 mass spectrometer (ABSciex). The protein sample was precipitated, digested by trypsin and 500 ng of digested peptides were injected in the mass spectrometer. Protein identifications were assigned using the Mascot algorithm (version 2.2, Matrix Science, London, UK) through the ProteinScape 3.1 package (Bruker Daltonics, Leipzig, Germany). Searches were performed against the Swissprot database (January 2013 release), and the S. aureus taxonomy and identifications from Mascot were validated with a protein false discovery rate (FDR) < 1%.

Samples were prepared for mass-spectrometry analyses as described (44 (link)). Briefly, samples solubilized in Laemmli buffer were precipitated with 0.1 M ammonium acetate in 100% methanol. After a reduction-alkylation step (Dithiothreitol 5 mM, Iodoacetamide 10 mM), proteins were digested overnight with 1/25 (w/w) of sequencing-grade porcin trypsin (Promega). The peptide mixtures were resolubilized in water containing 0.1% FA (solvent A) before being injected on nanoLC–MS/MS (NanoLC-2DPlus system with nanoFlex ChiP module; Eksigent, ABSciex, Concord, Ontario, Canada), coupled to a TripleTOF 5600 mass spectrometer (ABSciex). Peptides were eluted from the C-18 analytical column (75 μm ID × 15 cm ChromXP; Eksigent) with a 5–40% gradient of acetonitrile (solvent B) for 90 min. Data were searched against a TAIR database containing the GFP-TOR sequence as well as decoy reverse sequences (TAIR10_pep_20101214). Peptides were identified with Mascot algorithm (version 2.2, Matrix Science, London, UK) through the ProteinScape 3.1 package (Bruker). They were validated with a minimum score of 30, a P-value <0.05 and proteins were validated respecting a false discovery rate FDR <1%.