of gentamicin eliminates all extracellular bacteria. After that time, the cells were washed twice with PBS, overlaid with DMEM plus gentamicin (20 ug/ml final concentration), and incubated at 37uC with 5% CO2 for the remainder of the experiment. Macrophage cells were lysed with Triton X-100 for analysis at 1.5 h and 18 h post infection. Colony-forming units (cfu) counts were carried out to evaluate the survival and replication of intracellular bacteria. Briefly, at 1.5 h and 18 h time points, infected cells were washed three times with PBS and lysed by 1% Triton X-100 in PBS. The lysates were collected serially 10-fold diluted, and plated on LB agar plates, and the number of surviving bacteria enumerated following overnight incubation. Intracellular survival assays were performed in triplicate; the value shown is the mean. Soluble Protein Preparation Cell pellets were resuspended in 100 mM NH4HCO3, pH 8.4 buffer and lysed by using 0.1 mM zirconia/silica beads in a 2.0-ml Cryovial with vigorous vortexing for a total of 3 min with cooling steps. The supernatant and subsequent washes were transferred from the beads into new Cryovials. The beads were repeatedly washed until the supernatant was clear. After protein concentra- tion was determined for the samples, urea and thiourea were added to final concentrations of 7 and 2 M, respectively. Following addition of DTT (5 mM), the samples were incubated at 60uC for 30 min. The samples were then diluted 10-fold with buffer, and CaCl2 was added (1 mM) followed by trypsin in a 1:50 trypsin:protein ratio. The samples were digested for 3 h at 37uC and subsequently cleaned using a C18 solid phase extraction (SPE) column (Supelco). Each 1-ml 100- or 50-mg SPE column was conditioned with MeOH and rinsed with 0.1% TFA in water. Samples were introduced to the columns and then washed with 95:5 H2O:ACN that contained 0.1% TFA. Excess liquid was removed from the columns under vacuum, and the samples were eluted with 80:20 ACN:H2O that contained 0.1% TFA and concentrated in a SpeedVac (Thermo-Savant) to a final volume of ,100 ml. A BCA protein assay was performed to determine peptide concentrations prior to analysis. Insoluble Protein Preparation Cell pellets were treated and lysed as described above for soluble protein preparations. The lysate was centrifuged at 1,3006g at 4uC for 2 min, and the supernatant was transferred to polycar- bonate ultracentrifuge tubes (Beckman) and centrifuged at 4uC at 356,0006g for 10 min. Pellets were resuspended in 50 mM NH4HCO3, pH 7.8, and ultracentrifuged under the same conditions as used in the previous step. A BCA protein assay was performed on the pellets resuspended in water, and the samples were ultracentrifuged once again (as described above) before discarding the supernatant. Pellets were resuspended in ,200 mL of a solubilization solution (7 M urea, 2 M thiourea, 1% CHAPS in 50 mM ammonium bicarbonate, pH 7.8), and DTT was added to a final concentration of 9.7 mM. Samples were incubated at 60uC for 30 min, then diluted, and digested in the same manner as described for the global and soluble protein preparation. Samples were cleaned by using an appropriately sized strong cation exchange (SCX) SPE column (Supelco). Each 1-ml 100-mg column was conditioned with MeOH, rinsed in varying sequences and amounts with 10 mM ammonium formate in 25% ACN, pH 3.0; 500 mM ammonium formate in 25% ACN; and Nanopure water. Samples were acidified to a pH of #4.0 by adding 20% formic acid followed by centrifugation at 16,0006g for 5 min. Samples were then introduced to the columns and washed with 10 mM ammonium formate in 25% ACN, pH 3.0. Excess liquid was removed from the columns under vacuum. The Hfq, SmpB Targets in Salmonella samples were eluted with 80:15:5 MeOH:H2O:NH4OH and concentrated to ,100 ml using a SpeedVac. Final peptide concentrations were determined using a BCA protein assay. Capillary Liquid Chromatography (LC)-Mass Spectrometry (MS) Analysis LC-MS spectra were analyzed using the accurate mass and elution time (AMT) tag approach [51]. Briefly, the theoretical mass and the observed normalized elution time (NET) of each peptide identified by LC-MS/MS is used to construct a reference database of AMT tags, which serve as two-dimensional markers for identifying peptides in subsequent high resolution and high mass accuracy LC-MS analyses. A reference database of AMT tags for Salmonella Typhimurium has been generated through exhaustive SCX fractionation and LC-MS/MS analysis as described in our previous publications [52,53] and the LC-MS/ MS data from this experiment, obviating the need for further time consuming SCX LC-MS/MS analyses. This approach to proteomics research is enabled by a number of published and unpublished in-group developed tools, which are available for download at omics.pnl.gov [54,55,56,57,58]. Prior to the samples being analyzed they were subjected to a blocking and random- ization treatment to minimize the effects of systematic biases and ensure the even distribution of known and unknown confounding factors across the entire experimental dataset. Peptides from each of the soluble and insoluble protein preparations were separated by an automated in-house designed reverse-phase capillary HPLC system as described elsewhere [59]. Eluate from the HPLC was directly electrosprayed into an LTQ-Orbitrap mass spectrometer (LTQ-Orbitrap, Thermo Fisher Scientific, San Jose, CA) using electrospray ionization (ESI) with emitters described previously [60] and the ESI interface modified with an electrodynamic ion funnel [61]. Three biological replicates for each sample were analyzed on the ESI interface modified LTQ-Orbitrap mass spectrometer. Relevant information such as the elution time from the capillary LC column, the abundance of the signal (integrated area under the elution profile), and the monoisotopic mass (determined from charge state and the high accuracy m/z measurement) of each feature observed in the LTQ-Orbitrap was used to match the peptide identifications contained within the AMT tag database. An internally developed algorithm that calculates a probability of match using the mass, normalized elution time, peptide identification values from SEQUEST and their respective distributions was used to match features to peptide identifications [62]. Each peptide used for analysis was observed in at least one LC-MS analysis with a probability of a correct match being 0.9 and matches in the remaining LC-MS analyses were required to be a minimum probability of 0.5, with the additional constraint that at least three peptides with these constraints were required per protein identification. The abundances of these identified peptides are quantified using the chromatographic peak area and were used to infer the protein composition and abundance of the samples. The software program DAnTE [30] was employed to perform this abundance roll-up procedure to convert peptide information into protein information, thereby inferring relative protein abundances. Differences in protein expression between parent and iosgenic mutant was assessed via hypothesis testing, specifically a comprehensive ANOVA scheme included in the software program DAnTE. RNA preparation and microarray analysis Cell pellets from cell harvest above were resuspended in TE buffer pH8.0 containing Lysozyme and processed following the instructions of Qiagen RNeasy midi kit (Qiagen) in combination PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4809