In-solution trypsin digestion of the complex protein mixture was performed by the addition of trypsin at 1:25 for 5 h at 37°C followed by 1:50 digestion overnight. The tryptic digested samples were applied to SDS-PAGE to check for extensive digestion. Mass spectrometry analysis of tryptic peptides Methods for mass spectrometry (MS) analysis were previously described in detail [17]. Briefly, tryptic peptide digests (ca. 100 μg) were fractionated by 2D-LC-MS/MS, first using a Polysulfoethyl-A SCX column (4.6 × 50 mm, Nest Group, USA) followed by an Agilent 1100 series solvent delivery system (Agilent, Palo Alto, CA) online with a nano-electrospray LC-MS/MS system (LTQ-IT

mass spectrometer, Thermo-Finnigan, San Jose, CA). SCX fractions were delivered PF-02341066 research buy from 96-well plates onto a PicoTip microcapillary reversed-phase column (BioBasic C18, 75 μm × 10 cm, New Objective, Woburn, MA)

at a flow rate of 350 nL/min. Spectra were acquired in automated MS/MS mode with Selleckchem RO4929097 the top five parent ions selected for fragmentation using collision energy of 35%. LC-MS/MS was performed in three sequential m/z subscans (300-650, 650-900, 900-1500 m/z) to increase the sampling depth [16]. MS and MS/MS data from sequential runs were combined for search against the latest release of the S. dysenteriae Sd197 genome database in NCBInr using the Mascot search engine v.2.2 (Matrix Science, London, UK). This database contained 4502 protein sequences, including 231 proteins encoded by the two SD1 plasmids. Mascot search parameters allowed

for tryptic specificity of up to one missed cleavage, with methylthio-modifications of cysteine as a fixed modification and oxidation of methionine as a variable modification. The LTQ search parameters for +1, +2 and +3 ions included mass error tolerances of ± 1.4 Da for peptide ions and ± 0.5 Da for fragment ions. The false discovery rate (FDR) for peptide identifications was determined using the Mascot decoy database search option, with searches against a randomized S. dysenteriae Sd197 protein decoy database. Mascot search results of replicate 2D-LC-MS/MS experiments were further validated by estimating the FDR [19]via PeptideProphet™ and ProteinProphet™ Selleck ZD1839 [20] which are part of the Trans-Proteomic Pipeline (TPP) available at http://​tools.​proteomecenter.​org/​wiki/​index.​php?​title=​Software:​TPP. APEX quantitation of SD1 cell lysate LC-MS/MS datasets APEX quantitation of SD1 proteins was performed using the APEX Quantitative Proteomics Tool [21]v.1.1 as described previously [17]. Briefly, three steps were performed, building a SD1 training dataset, computing SD1 protein O i (expected number of unique proteotypic peptides for protein i) values, and calculating SD1 protein APEX abundances. Proteins in the training dataset were comprised of the 100 most abundant SD1 proteins based on high spectral counts per protein and high protein and peptide identification probabilities [22]. The training dataset.