Journal of Proteome Research Mass Spectrometry Data Set from Pathogen Portal Web Site The mass spectrometry data set “characterization of host and pathogen proteins aﬀected by Brucella abortus infection” was from Caprion Proteomics studies of B abortus, downloadable from pathogen portal web site hosted by proteomics resource center at http://www.pathogenportal.org/portal/portal/Path- Port/Home. Samples were processed for “intact bacteria” or “cell envelope” preparations from exponentially growing bacteria according to the Caprion protocol 24401 and 24302, as described before.23,35 Brieﬂy, to process intact bacteria for MS analysis, 50 μL aliquots of 20 μg protein samples were denatured in 50 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO) containing 8 M urea. After sonication, samples were centrifuged at 2000 g for 1 min and a 950 μL cold ( 20 C) chloroform/ methanol solution (2:1 v/v) were added. Samples were vortex mixed and incubated at 20 C for 2 h. Subsequently, 100 μL cold ( 20 C) methanol was added and samples were clariﬁed by centrifugation at 21 000 g for 10 min at 4 C. The super- natants were dried under vacuum, resuspended in 100 mM ammonium bicarbonate containing 8 M urea and 1% (w/v) of acid-labile surfactant (ALS; Waters, Milford, MA) and sonicated for 10 min. After centrifugation for 20 s at 2000 g, samples were incubated for 1 h at ambient temperature. A volume of 450 μL of 100 mM ammonium bicarbonate containing 5% (v/v) of aceto- nitrile was added. Lys-C (Wako, Richmond, VA) was added to yield a 1:50 enzyme to protein ratio. The samples were incubated for 3 h at 37 C. Trypsin (Promega, Madison, WI) was then added at a 1:50 enzyme to protein ratio and samples were incubated for an additional 16 h. Following proteolysis, TCEP (Pierce, Rockford, IL) was added to a ﬁnal concentration of 10 mM and samples were incubated for 30 min at ambient temperature and then lyophilized. To cleave the acid labile surfac- tant, samples were incubated for 30 min at ambient temperature in 100 μL of 1 N HCl and 100 μL of water were added into each sample with subsequent incubation for another half hour at ambient temperature. To process cell envelope for MS analysis, supernatants of exponentially growing B. abortus strains were clariﬁed, centrifuged at 100 000 g for 6 h at 4 C, pellets resuspended in double distilled water. The sample was sonicated, and 50 mM ammonium bicarbonate containing 2% acid-labile surfactant and 8 M urea were added. The sample was vortex mixed for 1 h. A sample of 50 μL of the OM suspension was added to a ﬁnal volume of 1 mL of a chloroform/methanol solution (2:1 v/v). The sample was vortex mixed and incubated at 20 C for 2 h. Subsequently, 100 μL of cold methanol ( 20 C) was added, and the sample was cleared by centrifugation for 10 min at 21 000 g. The supernatant was dried under vacuum and resuspended in 4 M urea; 50 mM ammonium bicarbonate pH 8.0. Lys-C was added at a 1:50 protein ratio. The samples were then diluted 4:1 with 50 mM ammonium bicarbonate buﬀer pH 8.0, and trypsin was added at a 1:25 protein ratio for an additional 16 h. Following proteolysis, the samples were distributed into 96-well plates for mass spectrometry analysis. Peptide digests were analyzed by liquid chromatography coupled to mass spectrometry (LC-MS) as described.23,35,36 The LC MS system consisted of a CapLC (Waters, Milford, MA) with a cooled autosampler and a QTOF Ultima (Waters, Milford, MA) controlled by MassLynx version 4.0 software. The peptide con- centrations were normalized and the samples were injected into a reversed-phase column (Jupiter C18, Phenomenex, Torrance, CA) for HPLC separation. Protein identiﬁcation was done by submitting LC MS spectra to Mascot software (MatrixScience, ARTICLE Boston, MA) for searching against the National Center for Biotechnology Information protein database (NCBI). The para- meters used for Mascot search and protein homology cluster- ing were previously detailed.23,35 Numbers of diﬀerent peptides for each identiﬁed protein were counted and listed in Table S4 (Supporting Information). Data Analysis Data analysis was performed using the R (http://www. r-project.org) and SAS (http://www.sas.com/) statistical software. It has been noted in literature that data derived from microarray platforms is heteroskedatic.37 39 To stabilize the variance,40,41 the vsn normalization method implemented as part of the Biocon- ductor suite (www.bioconductor.org) was applied to the quanti- ﬁed array intensities. In addition to removing heteroskedacity, this procedure corrects for nonspeciﬁc noise eﬀects by ﬁnding maximum likelihood shifting and scaling parameters for each array such that control probe variance is minimized. This calibration has been shown to be eﬀective on a number of platforms.42 Diﬀerentially reactive proteins between groups were deter- mined using a Bayes regularized t test adapted from Cyber-T for protein arrays,37,43 which has been shown to be more eﬀective than other diﬀerential expression techniques.44 To account for multiple comparison conditions, the Benjamini and Hochberg (BH) method was used to control the false discovery rate.45 After Benjamini and Hochberg correction, p-value smaller than 0.05 was considered signiﬁcant, and the corresponding protein was considered diﬀerentially reactive and serodiagnostic. Multiplex classiﬁers were constructed using linear and nonlinear Support Vector Machines (SVMs) using the “e1071” R package. Plots of receiver operating characteristic (ROC) curves were made with the “ROCR” R package. Sensitivity speciﬁcity and Area Under the Curve (AUC) were determined from the resulting ROC curves. Principle Component Analysis (PCA) to visually reduce and summarize signal intensity variance among the subjects was conducted in JMP software (www.JMP.com). Hierarchical clustering was used to group sera samples into subsets, such that those within each cluster (subset) are more closely related to one another than samples assigned to other clusters. Clustering is based on the degree of similarity between the protein intensity for each individual. MeV v4.6 (TM4Microarray Software Suite, www.tm4.org) was used to perform the clustering analysis. The following programs were utilized for computational pre- diction. TMHMM v2.0 was utilized for transmembrane domains prediction46 (http://www.cbs.dtu.dk/services/TMHMM/), SignalP v3.0 for signal peptide prediction47 (http://www.cbs.dtu.dk/ services/SignalP/), PSORTb v3.0. software for cellular location prediction48 (http://www.psort.org/psortb/). PI/MW tool from Swiss Institute of Bioinformatics was used to determine isolectric point (http://ca.expasy.org/tools/pi_tool.html). P value for enrichment statistical analysis was calculated using Fisher’s exact test in the R environment. ’ RESULTS Gene Amplification, Cloning and Protein Expression The 3198 predicted ORFs of B. melitensis (Bm) 16 M were subjected to ampliﬁcation from genomic DNA, and 1406 ORFs were cloned and printed on pilot chip.5 In this study, another 1640 ORFs were successfully cloned using our high throughput recombination method. About one-fourth of the cloned genes 4815 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824