Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer version 1.4. Charge state deconvolution and deisotoping was not performed. All MS/MS samples were analyzed using Mascot, Sequest (XCorr Only; Thermo Fisher Scientific, San Jose, CA, USA; version 1.3.0.339) and X! Tandem (GPM.org;
version CYCLONE (2010.12.01.1)) assuming digestion with trypsin. A custom E. coli database was generated by combining the fasta files from uniprot.org from the following E. coli strains: 12009/EHEC, 2009EL-2050, 2009EL-2071, Ipatasertib in vitro 2011C-3493, 11128/EHEC, O157:H7, EC4115/EHEC, TW14359/EHEC, and 11368/EHEC. This E. coli fasta file selleck screening library consists of 47,819 entries and was generated in May 2013. Mascot, Sequest (XCorr Only) and X! Tandem were searched with a fragment ion mass tolerance of 0.100 Da and a parent ion tolerance of 10.0 PPM; carbamidomethyl of cysteine and iTRAQ4plex of lysine and the n-terminus were specified as fixed modifications while deamidation of asparagine and glutamine, oxidation of methionine and iTRAQ4plex of tyrosine were specified as variable modifications. Scaffold (version Scaffold_4.0.6) was used to validate MS/MS based peptide and protein identifications,
as described above for ‘Bottom-up Proteomics’. The O157-proteome as expressed in LB was used as the reference against which all the other O157-proteomes were compared. Two biological replicate samples (Sample A and B), corresponding to the duplicate experiments described under ‘Culture conditions, selleckchem and processing for proteomics’ above, were analyzed separately. In addition, each sample was analyzed twice (Run A and Run B; technical replicates) to cover the entire spectra of proteins in these samples. Only proteins that were consistently identified were selected for analysis. Thiamet G Statistics and bioinformatics The Student t-Test (two-tailed) was used to evaluate differences between the means of the O157 optical densities and viable counts recovered from the different cultures and a values of p < 0.05 was considered significant. Putative
functions were determined by querying the Conserved Domain Database (CDD) at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, and associated metabolic pathways were determined using the KEGG pathway database at http://www.genome.jp/kegg/pathway.html. Cellular and sub-cellular locations of proteins were determined as described previously [17]. Results pH and VFA content The pH and VFA concentrations were comparable amongst all rumen fluid samples, indicating consistency in maintenance diet being fed and the ruminal chemistry between the two animals enrolled in the study (Tables 1 and 2). The pH of the uRF ranged from 6.4-6.7 at collection [28–31] but attained a more neutral pH after filtering, as seen with dRF (pH 7.4–7.9) and fRF (pH, 7.2–7.7) in both experiments (Tables 1 and 2).
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