Computational protocol: Dataset of the proteome of purified outer membrane vesicles from the human pathogen Aggregatibacter actinomycetemcomintans

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Protocol publication

[…] The analysis of the in-solution digest samples was achieved using LC-MS/MS (DDA, 5 MS/MS channels) with a Synapt G2 mass spectrometer (Waters, Sollentuna, Sweden) that was linked to a nano UPLC (Waters, Sollentuna, Sweden). Separation of the peptides was performed by C18 nano reversed phase chromatography (Acquity nano UPLC column 1.8 mm HSS T3 75 mm×200 mm). The peptides were separated at a flow rate of 300 nl/min using a 4 h long linear gradient (1–30 percent acetonitrile for 3 h, 30–50 percent acetonitrile for 1 h). Spectra were processed using the ProteinLynx Global Server 2.5.2 software (Waters, Sollentuna, Sweden) with lockspray calibration and fast de-isotoping for the MS and MS/MS mode. In addition, the spectra were also processed using the Mascot Distiller (version 2.4.3.3, Matrix Science, London, UK) and the standard settings for DDA data from Waters instruments. Database searches using the peaklist files of the processed mass spectra were performed using the Mascot search engine (version 2.4, MatrixScience, London, UK) in the database of A. actinomycetemcomitansserotype e strain SC1083, which is available at Ensembl Bacteria at the URL: http://bacteria.ensembl.org/aggregatibacter_actinomycetemcomitans_serotype_e_str_sc1083/Info/Index. The reason for using the database of another serotype e strain was that the genome of strain 173 is not available. The parameters for the database searches permitted mass errors of 20 ppm (MS mode) and 0.1 Da (MS/MS mode), respectively. Modifications included variable oxidation of methionine, N-terminal acetylation, deamidation (N,Q) and fixed cysteine derivation by carbamidomethylation. The false discovery rate was set to <1%. Compilation of non-redundant protein lists was carried out using the Protein Extractor of the ProteinScape server (version 3, Bruker Daltonik GmbH, Bremen, Germany). Ion scores of individual MS/MS spectra lower than 30 and Mascot protein scores lower than 100 were not considered for the compilation of the identified proteins. [...] The final list of identifications includes the proteins that are detected in at least three of the four OMV preparations, which were analyzed. This list contains 151 proteins, which are sorted according to their Clusters of Orthologous groups (COG) categories. The COG groups were created manually using the complete list of gene identifiers of the genome of strain SC1083 for batch searches in the COG database at National Center for Biotechnology Information (NCBI) at the URL: http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. The COG classifiers obtained by these searches were grouped according to the definitions provided in the NCBI conserved domains database . The subcellular locations of the identified proteins were predicted using the program PSORTb 3.0 , and members of KEGG pathways were identified using the KOBAS 2.0 server and the annotations of the A. actinomycetemcomitans strain D7S genome as a template. […]

Pipeline specifications

Software tools PLGS, Mascot Distiller, Mascot Server, PSORTb, KOBAS
Databases COGs KEGG
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Aggregatibacter actinomycetemcomitans, Homo sapiens
Diseases Endocarditis, Periodontitis, Atypical Hemolytic Uremic Syndrome