The LCQ was run in a top five configuration, with one MS scan and five MS/MS scans. Dynamic exclusion was set to 1 with a limit of 30 seconds. Peptide identifications were made using
SEQUEST (Thermo Finnigan) through the Bioworks Browser 3.2, as described previously [23]. Sequential database searches were performed using the O157 strains EDL933 and Sakai FASTA database from European Bioinformatics Institute http://www.ebi.ac.uk/newt/display using static carbamidomethyl-modified cysteines and differential oxidized methionines. These protein databases (Escherichia coli this website (strain Sakai/O157:H7/RIMD 0509952/EHEC) – Tax ID: 386585 and Escherichia coli (strain EDL933/ATCC 700927/O157:H7/EHEC) – Tax ID: 155864) have a total of 10,737entries. A reverse O157 strain EDL933 FASTA database was spiked in to provide noise and determine validity of the peptide hits, so that known
and theoretical MS-275 in vivo protein hits can be determined without compromising the statistical relevance of all the data [26]. The MS data was searched with a 2-Dalton window on the MS precursor with a 0.8 Dalton on the fragment ions. Peptide score JSH-23 research buy cutoff values were chosen at cross-correlation values (Xcorr) of 1.8 for singly charged ions, 2.5 for doubly charged ions, and 3.0 for triply charged ions, along with delta rank scoring preliminary cutoff (deltaCN) values of 0.1, and cross-correlation normalized values (RSp) of 1. The cross-correlation values chosen for each peptide assured a high confidence match for the different charge states, while the deltaCN values ensured the uniqueness of the peptide hit. The RSp value of 1 ensured that the peptide matched the top hit in the preliminary scoring. At these peptide filter values, very few reverse database hits were
observed, which permitted a higher confidence in the few single peptide protein identifications. Furthermore, single hit proteins were manually validated to ensure relevance. Bioinformatics Cellular location of proteins was determined using amino acid sequences of cognate proteins in the O157 sequence databases at http://www.ncbi.nlm.nih.gov/protein. In addition, extracytoplasmic proteins were verified for the presence of signal sequences using the GNAT2 program SignalP 3.0 at http://www.cbs.dtu.dk/services/SignalP, and subcellular localization of other proteins confirmed using the PSORT/PSORT-B program (http://psort.nibb.ac.jp/). Putative functions were determined by querying the Conserved Domain Database (CDD) at http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi Protein components of the O157 DMEM-proteome with adhesion potential were shortlisted using Vaxign, a reverse vaccinology based vaccine target prediction and analysis system at http://www.violinet.org that utilizes the SPAAN algorithm [27].