Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from those produced by nonspecific LPS stimulation. These patterns of differentially expressed proteins suggest mechanisms of pathogenesis as well as methods for diagnosis and monitoring Rickettsia infections. The genus Rickettsia contains non-motile, Gram-negative, obligately intracellular alphaproteobacteria that are of global medical and veterinary health importance due to their endemicity and re-emergence. From the clinical and antigenic perspectives, rickettsial diseases are classified into two groups, spotted fever and typhus. Nearly all of the numerous spotted fever group rickettsiae are transmitted by ticks. The most virulent ones are Rickettsia rickettsii, the agent of Rocky Mountain spotted fever, and Rickettsia conorii, the agent of Mediterranean spotted fever (boutonneuse fever), a disease prevalent throughout the Mediterranean, Africa, the Middle East, and India. In humans, the spotted fevers present as acute fever, headache, maculopapular rash, and vascular leakage that can lead to significant morbidity and mortality due to pulmonary and cerebral edema, particularly if there are delays in diagnosis and treatment (1.Walker D.H. Valbuena G.A. Olano J.P. Pathogenic mechanisms of diseases caused by Rickettsia.Ann. N.Y. Acad. Sci. 2003; 990: 1-11Crossref PubMed Scopus (122) Google Scholar). The characteristic leakage of intravascular fluid is a consequence of the specific tropism of rickettsiae for endothelial cells (1.Walker D.H. Valbuena G.A. Olano J.P. Pathogenic mechanisms of diseases caused by Rickettsia.Ann. N.Y. Acad. Sci. 2003; 990: 1-11Crossref PubMed Scopus (122) Google Scholar). Rickettsial organisms enter endothelial cells through a calcium-dependent zipper-like entry mechanism involving the actin cytoskeleton (2.Walker T.S. Winkler H.H. Penetration of cultured mouse fibroblasts (L cells) by Rickettsia prowazeki.Infect. 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Immun. 1993; 61: 1926-1935Crossref PubMed Google Scholar). Understanding the host response to Rickettsia infection has been advanced by the development of a standardized model of endothelial cell infection using primary human umbilical vein cells (HUVECs) 1The abbreviations used are:HUVEChuman umbilical vein endothelial cellFDRfalse discovery rateIPAIngenuity Pathway AnalysisPMplasma membraneSIDstable isotopic dilutionSRMselected reaction monitoringWCLwhole cell lysateSTATsignal transducer and activator of transcriptionNSAFnormalized spectral abundance factorSAFspectral abundance factorACNacetonitrileSISstable isotope standard. (10.Walker D.H. Firth W.T. Edgell C.J. Human endothelial cell culture plaques induced by Rickettsia rickettsii.Infect. Immun. 1982; 37: 301-306Crossref PubMed Google Scholar). In this model, infected endothelial cells have been shown to express cytokines, interferons, cell surface adhesion molecules such as E-selectin, VCAM-1, ICAM-1 (11.Sporn L.A. Lawrence S.O. Silverman D.J. Marder V.J. E-selectin-dependent neutrophil adhesion to Rickettsia rickettsii-infected endothelial cells.Blood. 1993; 81: 2406-2412Crossref PubMed Google Scholar, 12.Dignat-George F. Teysseire N. Mutin M. Bardin N. Lesaule G. Raoult D. Sampol J. Rickettsia conorii infection enhances vascular cell adhesion molecule-1- and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells.J. Infect. Dis. 1997; 175: 1142-1152Crossref PubMed Scopus (48) Google Scholar, 13.Damås J.K. Davi G. Jensenius M. Santilli F. Otterdal K. Ueland T. Flo T.H. Lien E. Espevik T. Frøland S.S. Vitale G. Raoult D. Aukrust P. Relative chemokine and adhesion molecule expression in Mediterranean spotted fever and African tick bite fever.J. Infect. 2009; 58: 68-75Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and αVβ3 integrin (14.Bechah Y. Capo C. Grau G. Raoult D. Mege J.L. Rickettsia prowazekii infection of endothelial cells increases leukocyte adhesion through αvβ3 integrin engagement.Clin. Microbiol. Infect. 2009; 15: 249-250Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar), and pro-coagulants (tissue factor and von Willebrand factor) (15.Clifton D.R. Rydkina E. Huyck H. Pryhuber G. Freeman R.S. Silverman D.J. Sahni S.K. Expression and secretion of chemotactic cytokines IL-8 and MCP-1 by human endothelial cells after Rickettsia rickettsii infection: regulation by nuclear transcription factor NF-κB.Int. J. Med. Microbiol. 2005; 295: 267-278Crossref PubMed Scopus (41) Google Scholar, 16.Sporn L.A. Haidaris P.J. Shi R.J. Nemerson Y. Silverman D.J. Marder V.J. Rickettsia rickettsii infection of cultured human endothelial cells induces tissue factor expression.Blood. 1994; 83: 1527-1534Crossref PubMed Google Scholar, 17.Sporn L.A. Marder V.J. Interleukin-1α production during Rickettsia rickettsii infection of cultured endothelial cells: potential role in autocrine cell stimulation.Infect. Immun. 1996; 64: 1609-1613Crossref PubMed Google Scholar). These endothelial cellular responses explain aspects of the pathobiology of natural infections, including microvascular hemorrhage, endothelial leakage, and multiorgan failure (1.Walker D.H. Valbuena G.A. Olano J.P. Pathogenic mechanisms of diseases caused by Rickettsia.Ann. N.Y. Acad. Sci. 2003; 990: 1-11Crossref PubMed Scopus (122) Google Scholar). human umbilical vein endothelial cell false discovery rate Ingenuity Pathway Analysis plasma membrane stable isotopic dilution selected reaction monitoring whole cell lysate signal transducer and activator of transcription normalized spectral abundance factor spectral abundance factor acetonitrile stable isotope standard. An integrated understanding of the endothelial cellular response is not yet available. To address this question, we have undertaken a study of the global endothelial cell proteomic response to infection with R. conorii using quantitative proteomic profiling of R. conorii-infected HUVECs, including the analysis of plasma membrane and secreted proteins within the Golgi apparatus. Our experimental design was to use trypsin-mediated exchange of stable isotopes of H2O to quantify differences in protein expression of primary HUVECs infected with R. conorii versus those stimulated with LPS alone to control for nonspecific inflammatory effects. In whole cellular lysates, we observed that R. conorii-infected endothelial cells significantly up-regulated the JAK-STAT signaling pathway. By contrast, analysis of PM and Golgi fractions revealed up-regulation of platelet adhesion proteins and down-regulation of integrin/cadherin components. To identify proteins secreted by R. conorii-infected HUVECs, soluble Golgi fractions were analyzed. Here, we observed significant induction of HLA and β2-microglobulin, providing insights into major histocompatibility complex (MHC)-I-mediated antigen processing, important in the host cytotoxic T cell response. Finally, 34 rickettsial proteins were identified with high confidence in the Golgi apparatus, PM, or secreted protein fractions. These studies advance the understanding of the endothelial response to R. conorii infection through up-regulation of IFN- and MHC class I antigen presentation pathways and implications of the secretome for the host response and diagnostics. Link sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin) was from Pierce (Thermo Scientific, San Jose, CA). NanoLinkTM streptavidin magnetic beads (0.8 μm) were from Solulink, San Diego, CA (catalog no. M-1002); protease inhibitor mixture was from Sigma, St. Louis, MO (catalog no. P8340). LPS was from Escherichia coli 0111:B4 (Sigma). Pools of HUVECs were established from individual human umbilical cords grown in supplemented EGM-2 medium (Lonza). The cells were subcultured when the monolayer became confluent two or three times per week. In this study, the cells were used between passages 3 and 4. For infection, 15 × 106 primary HUVECs in T175 flasks were infected in BSL-3 containment, and subsequently lysates were prepared 10 days later and were inactivated in accordance with University of Texas Medical Branch IBC-approved protocols. Cellular infection was verified by immunofluorescent microscopy using a rabbit polyclonal serum against R. conorii and anti-rabbit IgG conjugated to Alexa 594 (Life Technologies, inc.). HUVECs were stimulated with LPS (50 ng/ml) overnight as controls. R. conorii (Malish 7 strain) was obtained from the American Type Culture Collection (ATCC; Manassas, Va.; catalog no. VR-613). The stock, aliquots of a 10% suspension of infected yolk sac containing 4 × 106 pfu per ml, was stored at −70 °C. HUVECs were infected with R. conorii and subjected to whole cell lysate, plasma membrane, and Golgi fractionation. Experiments were replicated twice. For each quantitative LC-MS/MS analysis, samples were subjected to label swapping as described below. LPS-stimulated or R. conorii-infected HUVECs were washed three times with PBS (37 °C) containing calcium and magnesium. PM proteins were cross-linked 15 min in 10 ml of PBS with 10 μl of EZ-Link Sulfo-NHS-SS-Biotin stock solution (100 mg/ml, freshly prepared in DMSO) as described previously (18.Zhao Y. Zhang W. Kho Y. Zhao Y. Proteomic analysis of integral plasma membrane proteins.Anal. Chem. 2004; 76: 1817-1823Crossref PubMed Scopus (230) Google Scholar). Afterward, cross-linker was quenched by addition of 5 ml of lysine solution (1 mg/ml), washed in ice-cold wash buffer (250 mm sucrose, 10 mm Tris, pH 7.4), and resuspended in ice-cold homogenization buffer (250 mm sucrose, 10 mm Tris, pH 7.4, 1:100 dilution of protease inhibitor (Sigma P8340), 1 mm NaF, and 1 mm Na3VO4). The cells were concentrated by centrifugation (10 min at 800 × g at 4 °C), resuspended in homogenization buffer, and Dounce homogenized using 15 strokes of pestle A and 10 strokes of pestle B. The homogenate was then centrifuged (10 min at 1,000 × g, 4 °C), and membranes were captured by addition of suspended streptavidin magnetic beads (15 ml/T165 flask) followed by gentle mixing at 4 °C for 1 h and magnetic capture. The membrane-bound streptavidin beads were then washed with 1 m KCl (high salt wash) three times, followed by washing in 0.1 m Na2CO3, pH 11.5 (high-pH wash), and then ice-cold hypotonic buffer (10 mm HEPES, pH 7.5, 1.5 mm MgCl2, 10 mm KCl, 1:100 dilution of protease inhibitor mixture, 1 mm NaF, and 1 mm Na3VO4). The streptavidin beads were resuspended in twice their volume of 2× SDS sample buffer containing 100 mm dithiothreitol (DTT). After vortexing, beads were removed with a strong magnet, and the supernatant was saved. The proteins in the supernatant were separated by SDS-PAGE and visualized with Colloidal Blue (Life Technologies, Inc.). The gel in each lane was cut into small slices. The proteins were digested with trypsin in-gel as described previously. Briefly, the gel particles were destained in 1 ml of water/methanol solution (50:50, v/v) containing 25 mm NH4HCO3, pH 8.0, three times, changing the solution every 10 min. The destained gel was then washed in 1 ml of an acetic/methanol solution (acetic acid/methanol/water, 10:40:50, v/v/v) for 3 h, with the solution changed every 1 h. The resulting gel was soaked in 1 ml of water for 40 min, changing the solvent twice every 20 min. The gel was then transferred into a 0.5-ml microcentrifuge tube and dehydrated by soaking the gel in 100% acetonitrile (ACN) until it became opaque white. The solution was removed, and the gel was dried in a SpeedVac for 20–30 min. The dried gel was rehydrated with an adequate amount of trypsin digestion solution (10 ng of trypsin/μl in 50 mm NH4HCO3, pH 8.0). The digestion was carried out at 37 °C overnight. To extract tryptic digest, the gel was soaked in 40 μl of extraction solution (ACN/trifluoroacetic acid/water, 50:5:45, v/v/v) for 60 min with vortexing. The extraction solution was then carefully removed with a gel-loading pipette tip, and the extraction was repeated once. The extracts were pooled and dried with a SpeedVac. The tryptic peptides were used for trypsin-catalyzed 18O labeling. Golgi preparations were performed as described (19.Bell A.W. Ward M.A. Blackstock W.P. Freeman H.N. Choudhary J.S. Lewis A.P. Chotai D. Fazel A. Gushue J.N. Paiement J. Palcy S. Chevet E. Lafrenière-Roula M. Solari R. Thomas D. Y Rowley A. Bergeron J.J. Proteomics characterization of abundant Golgi membrane proteins.J. Biol. Chem. 2001; 276: 5152-5165Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). In brief, the flow-through of the magnetic bead separation from above was adjusted to a final concentration of 1.4 m sucrose by addition of 2.0 m sucrose and transferred to SW41 centrifuge tubes. Samples were overlaid with 4 ml of 1.2 m sucrose solution, topped off with 0.8 m sucrose solution, and spun at 38,000 rpm in an SW41Ti rotor (246,000 × g) for 90 min at 4 °C. Crude Golgi preparations were harvested from the 0.8/1.2 m sucrose interface. An aliquot was assayed for total protein concentration by the bicinchoninic acid (BCA) assay (Thermo Scientific). An equal volume of 1 m KCl was added to the crude Golgi preparation, incubated for 25 min with rotation at 4 °C, and an equal amount of ice-cold Dulbecco's modified PBS (D-PBS) added to each tube. The Golgi preparations were spun at 40,000 rpm (271,000 × g) at 4 °C. The membranes were then resuspended in 0.5 μl/μg of protein of crude extract using a 100 mm ammonium carbonate solution, pH 11.0. Membranes were removed by centrifugation at 40,000 rpm (271,000 × g) for 60 min at 4 °C. The soluble supernatants, representing the secreted proteins, were denatured by addition of an equal volume of 8 m guanidine HCl. Insoluble membranes were subjected to SDS-PAGE, and digested with trypsin in-gel as described above. 50 μg of protein were reduced with 10 mm DTT for 30 min at room temperature. Protein cysteinyl residues were alkylated with 30 mm iodoacetamide for 2 h at 37 °C. Each sample was diluted 1:10 with 100 mm ammonium bicarbonate and digested with 40 μg of trypsin overnight at 37 °C, and each tryptic peptide mixture was desalted with a Sep-Pak® C18 cartridge (Waters, Milford, MA) following the manufacturer's instructions. Peptides were eluted from the cartridge with 80% acetonitrile and completely dried using a Speedvac. The tryptic digest of each sample was divided into two parts of equal volume. One part was labeled with [18O]H2O, the other part remained unlabeled (16O-H2O). Peptide C-terminal 16O/18O labeling was performed as described previously (20.Starkey J.M. Zhao Y. Sadygov R.G. Haidacher S.J. Lejeune W.S. Dey N. Luxon B.A. Kane M.A. Napoli J.L. Denner L. Tilton R.G. Altered retinoic acid metabolism in diabetic mouse kidney identified by O isotopic labeling and 2D mass spectrometry.PLoS One. 2010; 5: e11095Crossref PubMed Scopus (40) Google Scholar, 21.Sadygov R.G. Zhao Y. Haidacher S.J. Starkey J.M. Tilton R.G. Denner L. Using power spectrum analysis to evaluate 18O-water labeling data acquired from low resolution mass spectrometers.J. Proteome Res. 2010; 9: 4306-4312Crossref PubMed Scopus (18) Google Scholar). The dried peptide samples were redissolved with 3 μl of anhydrous acetonitrile, 10 mg of immobilized trypsin (Applied Biosystems, CA), and 200 μl of normal water (H216O) or heavy water H218O containing 50 mm ammonium bicarbonate was added to the Rickettsia-infected and LPS control peptides, respectively, and both samples were incubated for 48 h at 37 °C. Supernatants were collected using a spin column and mixed as follows: 18O-labeled peptides from Rickettsia-treated sample mixed with 16O-labeled peptides from LPS particle-treated sample (forward labeling); 16O-labeled peptides from Rickettsia-treated sample mixed with 18O-labeled peptides from LPS particle-treated sample (reverse labeling). After mixing, the samples were desalted with a SepPak C18 cartridge (Waters). The desalted peptides were stored at −80 °C for LC-MS/MS analysis. Dried peptide samples were redissolved in 2 μl of acetonitrile and diluted with 40 μl of 0.1% formic acid. LC-MS/MS analysis was performed with a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) equipped with a nanospray source with an on-line Easy-nLC 1000 nano-HPLC system (Thermo Scientific, San Jose, CA). Ten microliters of each peptide solution were injected and separated on a reversed phase nano-HPLC C18 column (75 μm × 150 cm) with a linear gradient of 0–35% mobile phase B (0.1% formic acid, 90% acetonitrile) in mobile phase A (0.1% formic acid) over 120 min at 300 nl/min. The mass spectrometer was operated in the data-dependent acquisition mode with a resolution of 70,000 at full scan mode and 17,500 at MS/MS mode. The 10 most intense ions in each MS survey scan were automatically selected for MS/MS. The acquired MS/MS spectra were analyzed by 1.4 (22.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar) using default parameters (supplemental File 1) in the Swiss-Prot human protein databases (downloaded on February 2013, 20,247 protein entries) using a mass tolerance of ±20 ppm for precursor and product ions and a static mass modification on cysteinyl residues that corresponded to alkylation with iodoacetamide. Differential modifications were defined to be 18O-labeled C-terminal and oxidized methionine with a maximum of two missed cleavages. Protein identification data (accession numbers, peptides observed, sequence coverage) are in supplemental Tables 1–3. Annotated spectra of host proteins identified with single peptides are in supplemental file 2. Annotated spectra of rickettsial proteins identified with single peptides are in supplemental file 3. The FDR cutoff for peptide and protein identification is 0.01. For each subcellular fraction, the experiments were repeated after swapping the 18O-labeling between Rickettsia-infected cells and LPS-treated cells. This label swapping strategy allows for detection of irreproducible ratios that might arise due to interference in precursor quantification provided there is a minimum of two biological replicates. Each dataset was first centered by subtracting the most frequent value in that dataset and then subjected to MaxQuant Significant A analysis (22.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref PubMed Scopus (9223) Google Scholar). Next, the forward and reverse datasets were plotted with forward log2 heavy/light (H/L) ratio (x axis) against reverse log2 H/L ratio (y axis). Only the proteins that have a Significant A p value below 0.05 and also located in either upper-left or lower-right quadrant are considered to be the significantly expressed proteins. The normalized spectral abundance factor (NSAF) value for each protein was calculated as described (23.Zybailov B. Mosley A.L. Sardiu M.E. Coleman M.K. Florens L. Washburn M.P. Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae.J. Proteome Res. 2006; 5: 2339-2347Crossref PubMed Scopus (820) Google Scholar) in Equation 1, (NSAF)k=ILk∑i=1NILi(Eq. 1) where the total MS intensity (I) of the matching peptides from protein k was divided by the protein length (L) and then divided by the sum of I/L for all uniquely identified proteins in the dataset. The SID-SRM-MS assays were developed as described previously (24.Zhao Y. Brasier A.R. Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways.Methods. 2013; 61: 313-322Crossref PubMed Scopus (44) Google Scholar, 25.Zhao Y. Tian B. Edeh C.B. Brasier A.R. Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 1513-1529Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). For each targeted protein, two or three peptides were initially selected and then the sensitivity and selectivity of these were experimentally evaluated as described previously (24.Zhao Y. Brasier A.R. Applications of selected reaction monitoring (SRM)-mass spectrometry (MS) for quantitative measurement of signaling pathways.Methods. 2013; 61: 313-322Crossref PubMed Scopus (44) Google Scholar, 25.Zhao Y. Tian B. Edeh C.B. Brasier A.R. Quantitation of the dynamic profiles of the innate immune response using multiplex selected reaction monitoring-mass spectrometry.Mol. Cell. Proteomics. 2013; 12: 1513-1529Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The peptide with the best sensitivity and selectivity was selected as the surrogate for that protein. For each peptide, 3–5 SRM transitions were monitored. The signature peptides and SRM parameters are listed in Table VI. The peptides were chemically synthesized incorporating isotopically labeled [13C615N4]arginine or [13C615N2]lysine to a 99% isotopic enrichment (Thermo Scientific). The amount of stable isotope standard (SIS) peptides was determined by amino acid analysis. The tryptic digests were then reconstituted in 30 μl of 5% formic acid, 0.01% TFA. An aliquot of 10 μl of 50 fmol/μl diluted SIS peptides was added to each tryptic digest. These samples were desalted with a ZipTip C18 cartridge. The peptides were eluted with 80% ACN and dried. The peptides were reconstituted in 30 μl of 5% formic acid, 0.01% TFA and were directly analyzed by LC-SRM-MS. LC-SRM-MS analysis was performed with a TSQ Vantage triple quadrupole mass spectrometer equipped with nanospray source (Thermo Scientific, San Jose, CA). 8–10 targeted proteins were analyzed in a single LC-SRM run. The on-line chromatography was performed using an Eksigent NanoLC-2D HPLC system (AB SCIEX, Dublin, CA). An aliquot of 10 μl of each of the tryptic digests was injected on a C18 reverse-phase nano-HPLC column (PicoFritTM, 75 μm × 10 cm; tip inner diameter of 15 μm) at a flow rate of 500 nl/min over 20 min in 98% buffer A (0.1% formic acid), followed by a 15-min linear gradient from 2 to 30% mobile buffer B (0.1% formic acid, 90% acetonitrile). The TSQ Vantage was operated in high resolution SRM mode with Q1 and Q3 set to 0.2 and 0.7-Da full width half-maximum. All acquisition methods used the following parameters: 2100 V ion spray voltage, a 275 °C ion-transferring tube temperature, and a collision-activated dissociation pressure at 1.5 millitorr. The S-lens voltage used corresponded to the value in S-lens table generated during MS calibration.Table VISID-SRM-MS assays for human and rickettsial proteins All SRM data were manually inspected to ensure peak detection and accurate integration. The chromatographic retention time and the relative product ion intensities of the analyte peptides were compared with those of the SIS peptides. The variation of the retention time between the analyte peptides and their SIS counterparts should be within 0.05 min, and the difference in the relative product ion intensities of the analyte peptides and SIS peptides were below 20%. The peak areas in the extract ion chromatography of the native and SIS version of each signature peptide were integrated using Xcalibur® 2.1. The default values for noise percentage and baseline subtraction window were used. The ratio between the peak area of native and SIS version of each peptide was calculated. High confidence protein identifications were subjected to pathway enrichment analysis using the Protein ANalysis THrough Evolutionary Relationship (Panther) pathway classification system (26.Mi H. Lazareva-Ulitsky B. Loo R. Kejariwal A. Vandergriff J. Rabkin S. Guo N. Muruganujan A. Doremieux O. Campbell M.J. Kitano H. Thomas P.D. The PANTHER database of pr