2D-LC/MS/MS analysis of Escherichia coli under optimal growth condition

2D-LC/MS/MS analysis was used to examine the proteome of E. coli O157:H7 strain Sakai during exponential growth under optimal condition (i.e., from 35°C aw 0.993). MS/MS data obtained from each protein sample were processed by the Computational Proteomics Analysis System (CPAS), a web-based system built on the LabKey Server (v9.1, released 02.04.2009). The experimental mass spectra produced were subjected to a semi-tryptic search against the combined databases of E. coli O157:H7 Sakai (5,318 entries in total) downloaded from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, downloaded 25.11.2008) using X!Tandem v2007.07.01. These databases included the E. coli O157:H7 Sakai database (5230 entries, NC_002695.fasta) and two E. coli O157:H7 Sakai plasmid databases, plasmid pO157 (85 entries, NC_002128.fasta) and plasmid pOSAK1 (three entries, NC_002127.fasta). The parameters for the database search were as follows: mass tolerance for precursor and fragment ions: 10 ppm and 0.5 Da, respectively; fixed modification: cysteine cabamidomethylation (+57 Da); and no variable modifications. The search results were then analyzed using the PeptideProphet and ProteinProphet algorithms from the Trans Proteomic Pipeline v3.4.2. All peptide and protein identifications were accepted at PeptideProphet and ProteinProphet of ≥0.9, corresponding to a theoretical error rate of ≤2%.