Computational protocol: Characterisation of Pellicles Formed by Acinetobacter baumannii at the Air-Liquid Interface

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Protocol publication

[…] Purified matrixes were treated with 0.02% cellulase (Sigma-Aldrich, Lyon, France) at 37°C overnight with shaking. Samples were separated by discontinuous SDS-polyacrylamide gel electrophoresis (4.5% stacking, 15% separating gel) and gels were stained with 0.1% (wt/vol) Coomassie brilliant blue G 250 solution. Excised bands were washed several times with water and dried for 2 hours. Trypsin digestion was performed as described by Marti et al. . Identification of secreted proteins was performed by LC-MS/MS. All experiments were done on a LTQ-Orbitrap Elite (Thermo Scientific) coupled with an Easy nano-Liquid Chromatography II system (Thermo Scientific). Tryptic peptides were eluted from 2 to 55% of B solution (0.1% formic acid in acetonitrile) over 14 min, from 55 to 100% of B solution in 1 min and 10 min at 100% of B solution. The samples were analysed using the CID (Top20) method. Raw data files were processed using Proteome Discoverer 1.3 software (Thermo Scientific). Peak lists were searched using the MASCOT search engine (Matrix Science) against the database A. baumannii ATCC17978 protein sequences at http://www.ncbi.nlm.nih.gov. Database searches were performed with the following parameters: 2 missed trypsin cleavage sites allowed; variable modifications: carbamidomethylation of cystein, oxidation of methionine. Quantitation was based on label-free approach using spectral counting (peptide spectral match – PSM). Protein relative abundance was expressed after minimum-maximum normalization method. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server
Application MS-based untargeted proteomics
Organisms Acinetobacter baumannii, Escherichia coli
Chemicals Glucose