Est was used for statistical significance analysis of spot abundance changes. It is a non-parametric two sample distribution-free t-test and assesses whether two independent samples of observations come from the same distribution:U n1nn1(n11)R1 , where n1 and n2 are num-bers of observations in the samples and R1 is the sum of the ranks of the observations in sample 1. P-values determined by this test are based on 3 n 5 observations, which reflect 2D spot intensity data from an equal number of replicate gels. Provided that spot abundance ratios were 1.5, p-values < 0.02 were considered statistically significant. Protein spots observed in only one the two sample groups required spot matches among at least three technical replicates, but p-values could not be determined.Nearly all spots derived from 2D gels of the three Y. pestis subcellular fractions were analyzed by mass spectrometry (MS) two or more times. This was necessary in order to link each spot abundance change unambiguously to identification of a distinct protein; limitation of spot resolution in 2D gels is a known problem when the analyzed samples are highly complex. Prerequisites for confident spot identification were known protein identities of surrounding spots with equal or higher abundance and the comparison of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28893839 Mascot scores in those spots. Methods for spot cutting and protein digestion with trypsin were reported previously [45]. Peptide digests were analyzed using a MALDITOFTOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems) and a nano-electrospray LC-MS/MS system (LTQ ion trap mass spectrometer, Thermo-Finnigan, San Jose, CA) equipped with an Brefeldin A side effects Agilent 1100 series solvent delivery system (Agilent, Palo Alto, CA). Reversed phase peptide separations for LC-MS/MS analysis were performed at nanoflow rates (350 nL/min). Technical details of MS and MS2 analysis methods have been described [45]. The data were searched against the latest release of the Y. pestis KIM strain subset of the NCBInr database, using the Mascot searching engine v.2.1 (Matrix Science, London, UK). Carbamidomethyl was invariably selected as a fixed modification and one missed tryptic cleavage was allowed. MALDI search parameters (+1 ions) included mass error tolerances of ?100 ppm for peptide precursor ions and ?0.2 Da for fragment ions. LTQ ion trap search parameters (+1, +2 and +3 ions) included mass error tolerances of ?1.4 Da for peptide precursor ions and ?0.5 Da for fragment ions. Protein identifications were buy Avasimibe accepted as significant when Mascot protein scores >75 were obtained. Using a randomized decoy database, setting a default significance threshold of 0.05 in the Mascot algorithm and requiring two peptide e-values < 0.1 per protein identification, the false positive rate of proteins by LC-MS/MS was estimated to be <0.5 . Bioinformatic predictions of Y. pestis KIM iron transporters and binding proteins, of transmembrane domains, of protein export signal motifs and of b-barrel OM protein motifs were derived from the algorithms utilized in TransportDB http://www.membranetransport. org, TMHMM and SignalP http://www.cbs.dtu.dk/services and PRED-TMBB [46], respectively.ResultsUsing subcellular fractionation and differential 2D gel display to assess the response of Y. pestis to iron starvationThree subcellular fractions of the Y. pestis strain KIM6+, a periplasmic, a cytoplasmic and a membrane fractionPieper et al. BMC Microbiology 2010, 10:30 http://www.biomedcentral.com/1471-2180/10.Est was used for statistical significance analysis of spot abundance changes. It is a non-parametric two sample distribution-free t-test and assesses whether two independent samples of observations come from the same distribution:U n1nn1(n11)R1 , where n1 and n2 are num-bers of observations in the samples and R1 is the sum of the ranks of the observations in sample 1. P-values determined by this test are based on 3 n 5 observations, which reflect 2D spot intensity data from an equal number of replicate gels. Provided that spot abundance ratios were 1.5, p-values < 0.02 were considered statistically significant. Protein spots observed in only one the two sample groups required spot matches among at least three technical replicates, but p-values could not be determined.Nearly all spots derived from 2D gels of the three Y. pestis subcellular fractions were analyzed by mass spectrometry (MS) two or more times. This was necessary in order to link each spot abundance change unambiguously to identification of a distinct protein; limitation of spot resolution in 2D gels is a known problem when the analyzed samples are highly complex. Prerequisites for confident spot identification were known protein identities of surrounding spots with equal or higher abundance and the comparison of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28893839 Mascot scores in those spots. Methods for spot cutting and protein digestion with trypsin were reported previously [45]. Peptide digests were analyzed using a MALDITOFTOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystems) and a nano-electrospray LC-MS/MS system (LTQ ion trap mass spectrometer, Thermo-Finnigan, San Jose, CA) equipped with an Agilent 1100 series solvent delivery system (Agilent, Palo Alto, CA). Reversed phase peptide separations for LC-MS/MS analysis were performed at nanoflow rates (350 nL/min). Technical details of MS and MS2 analysis methods have been described [45]. The data were searched against the latest release of the Y. pestis KIM strain subset of the NCBInr database, using the Mascot searching engine v.2.1 (Matrix Science, London, UK). Carbamidomethyl was invariably selected as a fixed modification and one missed tryptic cleavage was allowed. MALDI search parameters (+1 ions) included mass error tolerances of ?100 ppm for peptide precursor ions and ?0.2 Da for fragment ions. LTQ ion trap search parameters (+1, +2 and +3 ions) included mass error tolerances of ?1.4 Da for peptide precursor ions and ?0.5 Da for fragment ions. Protein identifications were accepted as significant when Mascot protein scores >75 were obtained. Using a randomized decoy database, setting a default significance threshold of 0.05 in the Mascot algorithm and requiring two peptide e-values < 0.1 per protein identification, the false positive rate of proteins by LC-MS/MS was estimated to be <0.5 . Bioinformatic predictions of Y. pestis KIM iron transporters and binding proteins, of transmembrane domains, of protein export signal motifs and of b-barrel OM protein motifs were derived from the algorithms utilized in TransportDB http://www.membranetransport. org, TMHMM and SignalP http://www.cbs.dtu.dk/services and PRED-TMBB [46], respectively.ResultsUsing subcellular fractionation and differential 2D gel display to assess the response of Y. pestis to iron starvationThree subcellular fractions of the Y. pestis strain KIM6+, a periplasmic, a cytoplasmic and a membrane fractionPieper et al. BMC Microbiology 2010, 10:30 http://www.biomedcentral.com/1471-2180/10.
