Dataset Information

Sphingomonas wittichii RW1 shotgun proteomics

ABSTRACT: Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence has indicated the existence of a tremendous potential for aromatic compound transformation, with 56 ring-hydroxylating dioxygenase subunits, 34 extradiol dioxygenases, and 40 hydrolases. To further characterize this enzymatic arsenal, new methodological approaches should be employed. Here, a large shotgun proteomic survey has been performed on cells grown on dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin, and compared to growth on acetate. Changes in the proteome were monitored over time. Peak lists were generated with the Mascot Daemon software (version 2.3.2; Matrix Science, London, UK) using the extract_msn.exe data import filter (Thermo Fisher Scientific) from the Xcalibur FT package (version 2.0.7; Thermo Fisher Scientific). Data import filter options were set to 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans) and 1000 (threshold). The mgf files from technical triplicates were merged, and MS/MS spectra were assigned using the Mascot Daemon 2.3.2 (Matrix Science) and a database containing the nonredundant RefSeq protein entries for S. wittichii RW1 (NCBI Taxonomy ID: 392499), comprising 5345 protein sequences totalling 1 800 684 amino acids. The search was performed using the following criteria: tryptic peptides with a maximum of two miscleavages, mass tolerances of 5 ppm on the parent ion and 0.5 Da on the MS/MS, fixed modification for carbamidomethylated cysteine, and variable modification for methionine oxidation. Mascot results were parsed using the IRMa 1.28.0 software. Peptides were identified with a P-value threshold below 0.05. Proteins were considered validated when at least two distinct peptides were detected. Using a selection of 11 result files and the appropriate decoy database, the false discovery rate for protein identification was estimated to be 0.33% with these parameters.

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Sphingomonas Wittichii

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Jean ARMENGAUD

PROVIDER: PXD000403 | Pride | 2013-10-22

REPOSITORIES: Pride

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Publications

Shotgun proteomics suggests involvement of additional enzymes in dioxin degradation by Sphingomonas wittichii RW1.

Hartmann Erica M EM Armengaud Jean J

Environmental microbiology 20130930 1

Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence indicated the existence of a tremendous potential for aromatic compound transformation, with 5 ...[more]

PMID: 24118890

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Project description:Nucleoids have been purified from Deinococcus deserti and Deinococcus radiodurans cells subjected to irradiation and short recovery (six replicates for each strain). The nucleoids have been analyzed by shotgun proteomics as described in Toueille M et al (2012) J Proteomics. Comparative proteomics pointed at specific proteins recruited at the nucleoids during the recovery stage after DNA damages produced by the irradiation. Data processing and bioinformatics: Peak lists were generated with the MASCOT DAEMON software (version 2.2.2) from Matrix Science using the extract_msn.exe data import filter (ThermoFisher) from the Xcalibur FT package (version 2.0.7) from ThermoFisher. Data import filter options were set at: 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold). Using the MASCOT search engine (version 2.2.04) from Matrix Science, we searched all MS/MS spectra against in-house polypeptide sequence databases. For Deinococcus radiodurans MS/MS assignments, the database contained i) the sequence of all currently annotated proteins coded by D. radiodurans BAA-816 genome (2629 proteins from chromosome 1 (NC_001263), 368 proteins from chromosome 2 (NC_001264), 130 proteins from plasmid MP1 (NC_000958), and 35 proteins from plasmid CP1 (NC_000958)), ii) 28 manual protein sequence curations from D. radiodurans R1, and iii) 121 additional ORF sequences predicted by the CONSORF consensus prediction system.This database thus comprises 3,311 polypeptide sequences, totaling 1,006,757 amino acids. For Deinococcus deserti MS/MS assignments, the database contained the sequence of all annotated proteins coded by D. deserti VCD115 chromosome and plasmids. This database comprises 3,455 polypeptide sequences, totaling 1,083,334 amino acids. Searches for tryptic peptides were performed with the following parameters: full-trypsin specificity, a mass tolerance of 10 ppm on the parent ion and 0.5 Da on the MS/MS, static modifications of carboxyamidomethylated Cys (+57.0215), and dynamic modifications of oxidized Met (+15.9949). The maximum number of missed cleavages was set at 1. All peptide matches with a peptide score below a stringent P value of 0.001 were filtered by the IRMa 1.18.1 parser. A protein was considered validated when at least two different peptides were detected in the same experiment. False-positives rate for protein identification was estimated using the appropriate decoy database as below 0.1% with these parameters.

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Dataset Information

Sphingomonas wittichii RW1 shotgun proteomics

Publications

Shotgun proteomics suggests involvement of additional enzymes in dioxin degradation by Sphingomonas wittichii RW1.

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