Lab on a chip technology

Spots of interest were excised from 2D gels and washed several times with water and dried for a few minutes. Trypsin digestion was performed overnight with a dedicated automated system (MultiPROBE II, Perkin-Elmer). The gel fragments were subsequently incubated twice for 15 min in a 0.1% CH₃CN solution in water to allow extraction of peptides from the gel pieces. Peptide extracts were then dried and dissolved in starting buffer for chromatographic elution, consisting of 3% CH₃CN and 0.1% HCOOH in water. Peptides were enriched and separated using lab–on–a–chip technology (Agilent, Massy, France) and fragmented using an on–line XCT mass spectrometer (Agilent). The ESI LC–MS/MS data were converted into DTA– format files that were further searched for proteins with MASCOT Daemon (Matrix Science [40] ). For protein identification, two strategies were employed to mine the maximum data. Measured peptides were searched in the NCBI nr–protein sequence database, viridiplantae (green plants [41] ), and in the Brassica EST database (Brassica Genome Gateway 2007, [42] ). Proteins with two or more unique peptides matching the protein sequence with a score >53, as defined by MASCOT, were considered as a positive identification. The spectra of each peptide were verified manually. In cases where protein identification data were lacking, BLAST analysis was performed [43] .

The peptides were enriched and separated using a lab-on-a-chip technology (Agilent, Massy, France) and fragmented using an on-line XCT mass spectrometer (Agilent). The fragmentation data were interpreted using the Data Analysis program (version 3.4, Bruker Daltonic, Billerica, MA, United States). For the protein identification, the MS/MS peak lists were extracted, converted into mgf-format files and compared to the C. jejuni, strain 81–176 protein database (UniprotKB, CP000538 for the chromosome, CP000549 for plasmid pTet and CP000550 for plasmid pVir) with the MASCOT Daemon search engine (version 2.6.0; Matrix Science, London, United Kingdom). The following search parameters were used: trypsin was used as the cutting enzyme, the mass tolerance for monoisotopic peptide window was set to ±1.0 Da and the MS/MS tolerance window was set to ±0.5 Da. Two missed cleavages were allowed. Carbamidomethylation, oxidized methionine, acetylation and pyroglutamate in Nt and amidation in Ct were chosen as variable modifications. Generally, the peptides with individual ions scores higher than the score indicated for p < 0.05 were selected. The proteins with two or more unique peptides matching the protein sequence were automatically considered as a positive identification. The main raw data are presented in Supplementary Data Sheet S1. Other raw data are available upon request.