Proteomics-driven Antigen Discovery for Development of Vaccines Against Gonorrhea
2016; Elsevier BV; Volume: 15; Issue: 7 Linguagem: Inglês
10.1074/mcp.m116.058800
ISSN1535-9484
AutoresRyszard A. Zielke, Igor H. Wierzbicki, Benjamin I. Baarda, Philip R. Gafken, Olusegun O. Soge, King K. Holmes, Ann E. Jerse, Magnus Unemo, Aleksandra E. Sikora,
Tópico(s)Reproductive tract infections research
ResumoExpanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform—isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry—to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens. Expanding efforts to develop preventive gonorrhea vaccines is critical because of the dire possibility of untreatable gonococcal infections. Reverse vaccinology, which includes genome and proteome mining, has proven very successful in the discovery of vaccine candidates against many pathogenic bacteria. However, progress with this approach for a gonorrhea vaccine remains in its infancy. Accordingly, we applied a comprehensive proteomic platform—isobaric tagging for absolute quantification coupled with two-dimensional liquid chromatography and mass spectrometry—to identify potential gonococcal vaccine antigens. Our previous analyses focused on cell envelopes and naturally released membrane vesicles derived from four different Neisseria gonorrhoeae strains. Here, we extended these studies to identify cell envelope proteins of N. gonorrhoeae that are ubiquitously expressed and specifically induced by physiologically relevant environmental stimuli: oxygen availability, iron deprivation, and the presence of human serum. Together, these studies enabled the identification of numerous potential gonorrhea vaccine targets. Initial characterization of five novel vaccine candidate antigens that were ubiquitously expressed under these different growth conditions demonstrated that homologs of BamA (NGO1801), LptD (NGO1715), and TamA (NGO1956), and two uncharacterized proteins, NGO2054 and NGO2139, were surface exposed, secreted via naturally released membrane vesicles, and elicited bactericidal antibodies that cross-reacted with a panel of temporally and geographically diverse isolates. In addition, analysis of polymorphisms at the nucleotide and amino acid levels showed that these vaccine candidates are highly conserved among N. gonorrhoeae strains. Finally, depletion of BamA caused a loss of N. gonorrhoeae viability, suggesting it may be an essential target. Together, our data strongly support the use of proteomics-driven discovery of potential vaccine targets as a sound approach for identifying promising gonococcal antigens. Despite current prevention and management strategies, infections caused by sexually transmitted pathogens affect hundreds of millions of women and men in both resource-constrained and developed countries (1Fernandez-Romero J.A. Deal C. Herold B.C. Schiller J. Patton D. Zydowsky T. Romano J. Petro C.D. Narasimhan M. Multipurpose prevention technologies: the future of HIV and STI protection.Trends Microbiol. 2015; 23: 429-436Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Gonorrhea remains a leading public health burden with an estimated 78 million new cases annually worldwide (2Newman L. Rowley J. Vander Hoorn S. Wijesooriya N.S. Unemo M. Low N. Stevens G. Gottlieb S. Kiarie J. Temmerman M. Global estimates of the prevalence and incidence of four curable sexually transmitted infections in 2012 based on systematic review and global reporting.PloS One. 2015; 10: e0143304Crossref PubMed Scopus (782) Google Scholar). Major challenges in eradicating this ancient human disease include increasing multidrug-resistance among Neisseria gonorrhoeae strains and the high incidence of asymptomatic infections that contributes to the spread of gonorrhea (3Lewis D.A. The Gonococcus fights back: is this time a knock out?.Sex. Transm. Infect. 2010; 86: 415-421Crossref PubMed Scopus (123) Google Scholar, 4Unemo M. Shafer W.M. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.Clin. Microbiol. Rev. 2014; 27: 587-613Crossref PubMed Scopus (479) Google Scholar). Gonococcal infections often have devastating sequelae in women including pelvic inflammatory disease, ectopic pregnancy, and infertility (5Edwards J.L. Apicella M.A. The molecular mechanisms used by Neisseria gonorrhoeae to initiate infection differ between men and women.Clin. Microbiol. Rev. 2004; 17: 965-981Crossref PubMed Scopus (190) Google Scholar, 6Westrom L.V. Sexually transmitted diseases and infertility.Sex. Transm. Dis. 1994; 21: S32-S37PubMed Google Scholar). In addition, gonorrhea during pregnancy causes chorioamnionitis, which can be complicated further by a septic abortion, premature membrane rupture, and preterm delivery. Infants born to mothers with cervical gonorrhea have an increased risk of neonatal gonococcal conjunctivitis, which can lead to corneal scarring and blindness. In men, untreated urethritis may evolve into penile edema, urethral stricture and epididymitis (7Woods C.R. Gonococcal infections in neonates and young children.Semin. Ped. Infect. Dis. 2005; 16: 258-270Crossref PubMed Scopus (64) Google Scholar, 8Campbell M.F. The surgical pathology of epididymitis.Ann. Surg. 1928; 88: 98-111Crossref PubMed Google Scholar). Furthermore, gonorrhea facilitates the transmission and acquisition of HIV (9Fleming D.T. Wasserheit J.N. From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection.Sex. Transm. Infect. 1999; 75: 3-17Crossref PubMed Google Scholar). For these reasons, it is critical to develop effective interventions against gonorrhea. Currently, strains resistant to the last effective option for empiric monotherapy—third-generation cephalosporins—are emerging and clinical treatment failures have been documented in several countries (4Unemo M. Shafer W.M. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.Clin. Microbiol. Rev. 2014; 27: 587-613Crossref PubMed Scopus (479) Google Scholar, 10Ohnishi M. Golparian D. Shimuta K. Saika T. Hoshina S. Iwasaku K. Nakayama S. Kitawaki J. Unemo M. Is Neisseria gonorrhoeae initiating a future era of untreatable gonorrhea?: detailed characterization of the first strain with high-level resistance to ceftriaxone.Antimicrob. Agents Chemother. 2011; 55: 3538-3545Crossref PubMed Scopus (438) Google Scholar). History indicates that the incremental development of antibiotic resistance in N. gonorrhoeae inevitably continues to challenge antibiotic therapy (4Unemo M. Shafer W.M. Antimicrobial resistance in Neisseria gonorrhoeae in the 21st century: past, evolution, and future.Clin. Microbiol. Rev. 2014; 27: 587-613Crossref PubMed Scopus (479) Google Scholar). The World Health Organization recently recognized the escalating problem of the spread of antimicrobial resistance in N. gonorrhoeae and highlighted the importance of novel approaches to identify alternative strategies for the treatment and prevention of N. gonorrhoeae infections, including a gonorrhea vaccine (11World Health Organization. (2012) Global action plan to control the spread and impact of antimicrobial resistance in Neisseria gonorrhoeae, .Google Scholar). Pioneered by Rappuoli and colleagues, reverse vaccinology—which initially included genome and later proteome mining—has proven to be very successful in the discovery of vaccine candidates against many pathogenic bacteria (12Seib K.L. Zhao X. Rappuoli R. Developing vaccines in the era of genomics: a decade of reverse vaccinology.Clin. Microbiol. Infect. 2012; 18: 109-116Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 13Adamczyk-Poplawska M. Markowicz S. Jagusztyn-Krynicka E.K. Proteomics for development of vaccine.J. Proteomics. 2011; 74: 2596-2616Crossref PubMed Scopus (32) Google Scholar, 14Heckels J.E. Williams J.N. The influence of genomics and proteomics on the development of potential vaccines against meningococcal infection.Genome Med. 2010; 2: 43Crossref PubMed Scopus (4) Google Scholar, 15Delany I. Rappuoli R. Seib K.L. Vaccines, reverse vaccinology, and bacterial pathogenesis.Perspectives Med. 2013; 3: a012476Google Scholar). In particular, these methodologies paved the way for the newly developed group B meningococcal vaccine, which was a formidable effort for many years. Among the 28 antigens that were discovered and that elicited bactericidal antibodies against group B meningococci in vitro were the Neisserial heparin-binding antigen (NHBA), factor H-binding protein (fHbp), and the Neisserial adhesin A (NadA). These three proteins are formulated as part of the Bexsero meningococcal group B vaccine approved by the European commission in 2013 and the United States in February of 2015 (12Seib K.L. Zhao X. Rappuoli R. Developing vaccines in the era of genomics: a decade of reverse vaccinology.Clin. Microbiol. Infect. 2012; 18: 109-116Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Delany I. Rappuoli R. Seib K.L. Vaccines, reverse vaccinology, and bacterial pathogenesis.Perspectives Med. 2013; 3: a012476Google Scholar, 16Jerse A.E. Bash M.C. Russell M.W. Vaccines against gonorrhea: current status and future challenges.Vaccine. 2014; 32: 1579-1587Crossref PubMed Scopus (68) Google Scholar). Unfortunately, N. gonorrhoeae homologs of these proteins are not suitable vaccine targets (17Hadad R. Jacobsson S. Pizza M. Rappuoli R. Fredlund H. Olcen P. Unemo M. Novel meningococcal 4CMenB vaccine antigens- prevalence and polymorphisms of the encoding genes in Neisseria gonorrhoeae.APMIS. 2012; 120: 750-760Crossref PubMed Scopus (25) Google Scholar). Further, in contrast to meningococcal vaccines, progress on gonococcal vaccines has been hampered primarily by the absence of a vaccine-targetable surface capsule, exceptional variability of several surface antigens, a poor understanding of protective responses, and until relatively recently, the lack of a small laboratory animal model to systematically test potential vaccine candidates (16Jerse A.E. Bash M.C. Russell M.W. Vaccines against gonorrhea: current status and future challenges.Vaccine. 2014; 32: 1579-1587Crossref PubMed Scopus (68) Google Scholar). However, recent potential breakthroughs—such as the availability of transgenic mice to alleviate some host restrictions, new insights into immunosuppression mechanisms used by N. gonorrhoeae, and growing evidence that induction of Th1 response may be critical for vaccine efficacy—justify re-visiting gonorrhea vaccine development and initiating a significant focus in this area (16Jerse A.E. Bash M.C. Russell M.W. 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Vaccines against gonorrhea: current status and future challenges.Vaccine. 2014; 32: 1579-1587Crossref PubMed Scopus (68) Google Scholar). However, this is a very limited repertoire considering that, during the development of the Bexsero vaccine, out of nearly 600 candidates selected by in silico analysis, 350 recombinant meningococcal proteins were successfully expressed in Escherichia coli and evaluated for their surface exposure and ability to induce bactericidal antibodies (12Seib K.L. Zhao X. Rappuoli R. Developing vaccines in the era of genomics: a decade of reverse vaccinology.Clin. Microbiol. Infect. 2012; 18: 109-116Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Thus, a comprehensive antigen discovery program would be exceedingly valuable toward making a gonorrhea vaccine a reality. We are approaching this goal by applying proteomics-driven reverse vaccinology. In our previous study, a quantitative proteome analysis of cell envelopes and naturally released membrane vesicles derived from well-recognized N. gonorrhoeae laboratory strains FA1090, F62, MS11, and 1291 revealed a myriad of novel proteins, including 21 predicted outer membrane proteins (21Zielke R.A. Wierzbicki I.H. Weber J.V. Gafken P.R. Sikora A.E. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.Mol. Cell. Proteomics. 2014; 13: 1299-1317Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To extend these studies, herein we report high-throughput profiling of the N. gonorrhoeae cell envelope to identify ubiquitously and differentially expressed proteins in response to environmental cues relevant to infection including different oxygen tensions (aerobic, anaerobic), iron deprivation, and the presence of normal human serum. In addition, a subset of five identified proteins was evaluated for the capacity to induce antibodies that recognize a collection of diverse clinical isolates and have bactericidal activity against serum resistant and serum sensitive N. gonorrhoeae strains. The following N. gonorrhoeae strains were used in this study: FA1090 (22Connell T.D. Black W.J. Kawula T.H. Barritt D.S. Dempsey J.A. Kverneland Jr., K. Stephenson A. Schepart B.S. Murphy G.L. Cannon J.G. Recombination among protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the protein II family.Mol. Microbiol. 1988; 2: 227-236Crossref PubMed Google Scholar), MS11 (23Meyer T.F. Mlawer N. So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement.Cell. 1982; 30: 45-52Abstract Full Text PDF PubMed Scopus (139) Google Scholar), 1291 (24Apicella M.A. Breen J.F. Gagliardi N.C. 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N. gonorrhoeae isolates were cultured from frozen stocks stored at −80 °C onto gonococcal base agar solid medium (GCB 1The abbreviations used are:GCBgonococcal base agar solid mediumCcytoplasmicCEcell envelopesCFUcolony-forming unitsCOGClusters of Orthologous GroupsGCBLgonococcal base liquid mediumIPTGisopropyl-β-D-galactopyranosideiTRAQisobaric tagging for relative and absolute quantificationMVsmembrane vesiclesNHSnormal human serum2D-LCtwo-dimensional liquid chromatographySCXstrong cation exchangeSGCstandard growth conditionsSSsoluble fractions of supernatants., Difco, Sparks, MD) containing Kellogg's supplements I (1:100) and II (1:1000) (30Spence J.M. Wright L. Clark V.L. Laboratory maintenance of Neisseria gonorrhoeae.Curr. Protoc. Microbiol. 2008; (Chapter 4, Unit 4A 1)Crossref PubMed Scopus (18) Google Scholar). After incubation for 18–20 h at 37 °C in a humid, 5% CO2 atmosphere, transparent colonies with piliated or nonpiliated colony morphologies were sub-cultured onto GCB plates. Piliated N. gonorrhoeae variants were used for transformation, whereas nonpiliated bacteria were used in all other experiments. Following ∼18 h of incubation as described above, bacteria were harvested from the solid media using a polyester-tipped applicator (Puritan, Guilford, ME), and suspended to a final OD600 of 0.1 in gonococcal base liquid (GCBL) medium containing Kellogg's supplements I and II and sodium bicarbonate (at a final concentration of 0.042%) (30Spence J.M. Wright L. Clark V.L. Laboratory maintenance of Neisseria gonorrhoeae.Curr. Protoc. Microbiol. 2008; (Chapter 4, Unit 4A 1)Crossref PubMed Scopus (18) Google Scholar, 31Kellogg Jr., D.S. Peacock Jr., W.L. Deacon W.E. Brown L. Pirkle D.I. Neisseria gonorrhoeae. I. Virulence genetically linked to clonal variation.J. 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Evans T.M. Klimpel K.W. Induction and repression of outer membrane proteins by anaerobic growth of Neisseria gonorrhoeae.Infect. Immun. 1987; 55: 1359-1364Crossref PubMed Google Scholar). gonococcal base agar solid medium cytoplasmic cell envelopes colony-forming units Clusters of Orthologous Groups gonococcal base liquid medium isopropyl-β-D-galactopyranoside isobaric tagging for relative and absolute quantification membrane vesicles normal human serum two-dimensional liquid chromatography strong cation exchange standard growth conditions soluble fractions of supernatants. E. coli NEB5α and BL21(DE3) were used for genetic manipulations and as a host for heterologous protein expression, respectively. The E. coli strains were grown in Luria-Bertani (LB) medium (Difco) or maintained on LB agar plates at 37 °C supplemented with appropriate antibiotics. Antibiotics were used at the following concentrations: N. gonorrhoeae kanamycin (40 μg/ml) and erythromycin (0.5 μg/ml); E. coli kanamycin (50 μg/ml). For proteomic analysis of the N. gonorrhoeae cell envelope composition, N. gonorrhoeae FA1090 was first cultured on GCB, bacteria were collected from plates, suspended in GCBL to an OD600 of 0.1, and 100 μl aliquots were spread on solid media for simultaneous growth under aerobic (standard GCB, iron limited, and in the presence of human serum) and anaerobic (in the presence of nitrite) conditions, as described above. Bacteria were harvested when the colonies reached approximately similar size (after 18 and 36 h for aerobic and anaerobic conditions, respectively), and the cell envelope fractions were isolated as described previously (21Zielke R.A. Wierzbicki I.H. Weber J.V. Gafken P.R. Sikora A.E. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.Mol. Cell. Proteomics. 2014; 13: 1299-1317Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35Zielke R.A. Gafken P.R. Sikora A.E. Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.Curr. Protoc. Microbiol. 2014; 34 (1F 3 1–1F 3 16)Crossref Scopus (4) Google Scholar). Protein concentrations were measured using 2D Quant Kit (GE Healthcare, Piscataway, NJ). After isolation, the cell envelope-associated proteins were precipitated, trypsinized and labeled with iTRAQ reagents (AB Sciex, Waltham, MA) according to the procedures reported by Zielke et al. (21Zielke R.A. Wierzbicki I.H. Weber J.V. Gafken P.R. Sikora A.E. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.Mol. Cell. Proteomics. 2014; 13: 1299-1317Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35Zielke R.A. Gafken P.R. Sikora A.E. Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.Curr. Protoc. Microbiol. 2014; 34 (1F 3 1–1F 3 16)Crossref Scopus (4) Google Scholar). The following iTRAQ tags were used to label peptides in the cell envelope fractions derived from N. gonorrhoeae cultured on GCB under four growth conditions: 114 for standard aerobic; 115 for growth in the presence of 7.5% normal human serum; 116 for iron-limited; and 117 for anaerobic. iTRAQ-labeled peptides were separated using strong cation exchange (SCX) chromatography (21Zielke R.A. Wierzbicki I.H. Weber J.V. Gafken P.R. Sikora A.E. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.Mol. Cell. Proteomics. 2014; 13: 1299-1317Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35Zielke R.A. Gafken P.R. Sikora A.E. Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.Curr. Protoc. Microbiol. 2014; 34 (1F 3 1–1F 3 16)Crossref Scopus (4) Google Scholar). Desalted SCX fractions were analyzed by LC/ESI MS/MS with a ThermoScientific Easy-nLC II (Thermo Scientific) nano HPLC coupled to a hybrid Orbitrap Elite ETD (Thermo Scientific) mass spectrometer using an instrument configuration as described in (36Yi E.C. Lee H. Aebersold R. Goodlett D.R. A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation.Rapid Commun. In Mass Spectrom. 2003; 17: 2093-2098Crossref PubMed Google Scholar). In-line desalting was accomplished using a reversed-phase trap column (100 μm × 20 mm) packed with Magic C18AQ (5-μm, 200 Å resin; Michrom Bioresources, Auburn, CA), followed by peptide separations on a reversed-phase column (75 μm × 250 mm) packed with Magic C18AQ (5-μm, 100 Å resin; Michrom Bioresources) directly mounted on the electrospray ion source. A 90-min gradient from 7% to 35% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min was used for chromatographic separations. The heated capillary temperature was set to 300 °C and a spray voltage of 2250 V was applied to the electrospray tip. The Orbitrap Elite instrument was operated in the data-dependent mode, switching automatically between MS survey scans in the Orbitrap (AGC target value 1,000,000; resolution 60,000; and injection time 250 msec) and MS/MS scans in the OrbiTrap (AGC target value of 50,000; 15,000 resolution; and injection time 250 msec). The 15 most intense ions from the survey scan were selected for fragmentation by higher energy collisionally activated dissociation (HCD) with normalized collision energy of 40%. Selected ions were dynamically excluded for 45 s with a list size of 500 and exclusion mass-by-mass width ± 0.5. Data analysis was performed using Proteome Discoverer 1.4 (Thermo Scientific). The data were searched against a SwissProt N. gonorrhoeae FA1090 database with 1963 protein entries (downloaded on January, 17, 2012) that included common contaminants [the common Repository of Adventitious Proteins (cRAP)]. Trypsin was set as the enzyme with maximum missed cleavages set to 2. The precursor ion tolerance and the fragment ion tolerance were set to 10 ppm and 0.8 Da, respectively. Variable modifications included iTRAQ4Plex (+144.102 Da) on any N-Terminus, oxidation on methionine (+15.995 Da), methyl methanethiosulfonate on cysteine (+46.988 Da), and iTRAQ4Plex on lysine (+144.102 Da). Data were searched using Sequest HT. All search results were run through Percolator for scoring. Quantification was performed using the iTRAQ 4plex method built into Proteome Discoverer. The mass spectrometry data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository ProteomeXchange with the data set identifier PXD001944. Additionally, supplemental Table S2 lists all identified proteins with their accession number, a brief description, calculated score, protein coverage, number of unique and total peptides, iTRAQ ratios, molecular mass, and calculated pI. All statistical analyses were performed as we described previously (21Zielke R.A. Wierzbicki I.H. Weber J.V. Gafken P.R. Sikora A.E. Quantitative proteomics of the Neisseria gonorrhoeae cell envelope and membrane vesicles for the discovery of potential therapeutic targets.Mol. Cell. Proteomics. 2014; 13: 1299-1317Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 35Zielke R.A. Gafken P.R. Sikora A.E. Quantitative proteomic analysis of the cell envelopes and native membrane vesicles derived from gram-negative bacteria.Curr. Protoc. Microbiol. 2014; 34 (1F 3 1–1F 3 16)Crossref Scopus (4) Google Scholar, 37Sikora A.E. Zielke R.A. Lawrence D.A. Andrews P.C. Sandkvist M. Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases.J. Biol. Chem. 2011; 286: 16555-16566Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Briefly, only proteins identified with 1% false discovery rate (FDR) based on at least one unique peptide with ≥95% confidence were recorded. The default bias-correction was used and all quantitative variables were analyzed by the Proteome Discoverer 1.4. Subsequently, average ratios and standard deviations were calculated for proteins identified in three independent experiments. Proteins were considered ubiquitously expressed if the iTRAQ ratios were between 0.5 and 2, and were desig
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