Computational protocol: Identifying components required for OMP biogenesis as novel targets for antiinfective drugs

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Protocol publication

[…] Acquired MS spectra were processed with MaxQuant software package version 1.5.2.8 with integrated Andromeda search engine. Database search was performed against a target-decoy Ye serotype O:8 specific database obtained from Uniprot, containing 4172 protein entries, and 245 commonly observed contaminants. This database did not include an entry for TolC. Therefore, data were also analysed with a Ye Uniprot database containing 23,675 protein entries. Endoprotease trypsin was defined as the protease with a maximum missed cleavage of two. Oxidation of methionines and N-terminal acetylation were specified as variable modifications, whereas carbamidomethylation on cysteines was defined as a fixed modification. Initial maximum allowed mass tolerance was set to 4.5 ppm (for the survey scan) and 0.5 Da for CID fragment ions. A false discovery rate of 1% was applied at the peptide and protein level. The label-free algorithm was enabled, as was the “match between runs” option. MaxQuant output table was filtered for OM proteins (lipoproteins and ß-barrel proteins). Fourty-seven OM proteins could be identified and were kept for further analysis. Perseus software (version 1.5.0.15), a module from the MaxQuant suite, was used for calculation of the significance B (psigB) for each protein ratio (calculated based on averaged LFQ intensities) with respect to the distance of the median of the distribution of all protein ratios as well as its LFQ intensity. All proteins with psigB < 0.05 in a pairwise comparison were considered to be differentially expressed. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD004429 (http://proteomecentral.proteomexchange.org/cgi/GetDataset Username: [email protected] Password: XBxYXb4E) and in Table S4. Hydropathy indices of proteins were calculated using the tool http://www.gravy-calculator.de/. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Perseus, GRAVY Calculator
Databases ProteomeXchange
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Escherichia coli, Bacteria, Yersinia enterocolitica, Mus musculus
Diseases Bacterial Infections, Infection