The bacterial cells were grown on Colombia Blood Agar (CBA; Oxoid) for 18 h at 37 °C. Extracts were prepared from the second inoculum of each strain and each biological replicate was prepared independently on different day under identical growth conditions.
Extracted molecule
protein
Extraction protocol
Cell lysates were obtained by harvesting the growth of three plates of S. enterica and resuspending the cells in 20 mM Tris (pH=7.5). The cells were washed and collected by centrifugation at 5 100 ? g, 10 min. The resulting cell pellet was resuspended in 100 ?l of lysis solution containing 0.3% w/v SDS, 200 mM DTT and 50 mM Tris (pH=8.0), followed by the addition of 50 ?l of 0.5 M Tris (pH=8.0), 50 mM MgCl2, 1 mg ml-1 DNase 1 and 0.25 mg ml-1 RNase A. The samples were vortexed with 0.3 g of glass beads (? 105 ?m diameter) and homogenised five times for 1 min using the Mickle Cell Desintegrator (Mickle Laboratory Engineering) with 1 min cooling time on ice in between runs. The unbroken cells and cell debris were removed by centrifugation at 21 000 ? g for 30 min, 4?C, and the resulting supernatant was used for further analysis.
Label
na
Label protocol
na
Hybridization protocol
na
Scan protocol
na
Description
Rehydration and Isoelectric Focusing (IEF): Protein samples containing a total of 150 ?g of protein were mixed with rehydration buffer (7 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer pH 3-10 NL, (Amersham BioSciences)). The rehydration of the IPG strips was performed by the in-gel rehydration method (Rabilloud et al, 1994) using 18 cm IPG strips (pH 3-10 NL, Amersham BioSciences). The samples were left to rehydrate for 18 h at 20?C. IEF was performed using an Investigator 5000 (Genomic Solutions) power supply for 24 h, for a total of 85 000 Vh at a maximum voltage of 5000 V and a maximum current of 110 ?A.
Equilibration of the IPG strips and second dimension separation: The focused IPG strips were equilibrated in Tris/acetate equlibration buffer (Genomic Solutions) containing of 0.5% w/v SDS, 30% v/v glycerol and 6 M urea. The equilibration was performed in two consecutive steps of 30 min each. The first step was conducted in the presence of 50 mM DTT, and the second in the presence of 135 mM iodoacetamide. The second dimension separation was performed on 10% Duracryl ® gels (Proteomic Solutions). The gels were run for 5h, at a maximum voltage of 500 V and maximum power of 20 000 mW per gel using the Investigator 5000 power supply (Genomic Solutions). The gels were post-stained with fluorescent dye SYPRO Ruby (Molecular Probes) for 18 h on constant shaking and destained for 2 h in 10 % v/v methanol. The resulting 2D GE profiles were visualised using the Typhoon Scanner (Amersham Biosciences). To excise the spots the gels were double stained with Colloidal G and destained in 25 % v/v methanol.
Analysis of the 2D GE profiles: For the comparative analysis duplicate experimental samples from two different batches of each isolate were used (two technical replicates and two biological replicates for each isolate). The density/volume of the SYPRO Ruby-stained protein spots were determined using the software package Proteome Weaver (Definiens). For a protein to be reported as differentially expressed, the density values measured in Proteome Weaver were further subjected to the Student?s T-test, and only changes with p< 0.01 were considered significant. In gel trypsin digestion and peptide analysis using LC/MS/MS: In-gel digestion with trypsin was performed with a robotic digestion system (Investigator ProGest, Genomic Solutions).Colloidal G was removed by sequential washing with 50 mM ammonium hydrogen carbonate buffer and acetonitrile. Cysteine residues were reduced with DTT and derivatised by treatment with iodoacetamide. After further washing with ammonium hydrogen carbonate buffer, the gel pieces were again dehydrated with acetonitrile and dried at 60°C, prior to the addition of modified trypsin (Promega) (10 µl of a 6.5 ng µl-1 solution in 25 mM ammonium hydrogen carbonate). Digestion proceeded for 8 h at 37°C, and the products were recovered by sequential extractions with 25 mM ammonium hydrogen carbonate, 5% v/v formic acid, and acetonitrile. The pooled extracts were lyophilised, and redissolved in 0.1% v/v formic acid for mass spectrometry. Tandem electrospray mass spectra were recorded using a Q-TOF hybrid quadrupole / orthogonal acceleration time of flight spectrometer (Micromass) interfaced to a Micromass CapLC capillary chromatograph. Samples were dissolved in 0.1% v/v formic acid, and 6 µl was injected onto a Pepmap C18 trap column (300 µm ? 0.5 cm; LC Packings), and washed for 3 min with 0.1 % v/v formic acid. The flow rate was then reduced to 1 µl per min, and the peptides were eluted into the mass spectrometer with an acetonitrile / 0.1% v/v formic acid gradient (5% to 70 % v/v acetonitrile over 20 minutes). The capillary voltage was set to 3,500 V, and data-dependent MS/MS acquisitions were performed on precursors with charge states of 2, 3 or 4 over a survey mass range of 540-1200 Da. Known trypsin autolysis products and keratin-derived precursor ions were automatically excluded. The collision voltage was varied between 18 and 45 V depending on the charge and mass of the precursor. Product ion spectra were charge state de-encrypted and de-isotoped with a maximum entropy algorithm (MaxEnt 3, Micromass). Proteins were identified by correlation of uninterpreted tandem mass spectra to entries in SwissProt/TREMBL using ProteinLynx Global Server (Version 1.1, Micromass) and NCBInr using MASCOT. One missed cleavage per peptide was allowed, and an initial mass tolerance of 50 ppm was used in all searches. Cysteines were assumed to be carbamidomethylated, but other potential modifications were not considered in the first pass search. All matching spectra were reviewed manually, but in cases where the score reported by ProteinLynx global server was less than 100, additional searches were performed against the NCBI nr database using MASCOT, which utilises a robust probalistic scoring algorithm. Identifications based on a single matching peptide were verified manually using the MassLynx program Pepseq (Micromass).
Data processing
The density/volume of the SYPRO Ruby-stained protein spots were determined using the software package Proteome Weaver (Definiens). For a protein to be reported as differentially expressed, the density values measured in Proteome Weaver were further subjected to the Student?s T-test, and only changes with p< 0.01 were considered significant.
