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Carcinogenic Helicobacter pylori Strains Selectively Dysregulate the In Vivo Gastric Proteome, Which May Be Associated with Stomach Cancer Progression*

2018; Elsevier BV; Volume: 18; Issue: 2 Linguagem: Inglês

10.1074/mcp.ra118.001181

ISSN

1535-9484

Autores

Jennifer M. Noto, Kristie L. Rose, Amanda J. Hachey, Alberto G. Delgado, Judith Romero‐Gallo, Lydia E. Wroblewski, Barbara Schneider, Shailja C. Shah, Timothy L. Cover, Keith T. Wilson, Dawn A. Israel, Juan Carlos Roa, Kevin L. Schey, Yana Zavros, M. Blanca Piazuelo, Richard M. Peek,

Tópico(s)

Eosinophilic Esophagitis

Resumo

Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+ H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans. Helicobacter pylori is the strongest risk factor for gastric cancer. Initial interactions between H. pylori and its host originate at the microbial-gastric epithelial cell interface, and contact between H. pylori and gastric epithelium activates signaling pathways that drive oncogenesis. One microbial constituent that increases gastric cancer risk is the cag pathogenicity island, which encodes a type IV secretion system that translocates the effector protein, CagA, into host cells. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+ H. pylori strain, 7.13, recapitulates many features of H. pylori-induced gastric cancer in humans. Therefore, we sought to define gastric proteomic changes induced by H. pylori that are critical for initiation of the gastric carcinogenic cascade. Gastric cell scrapings were harvested from H. pylori-infected and uninfected gerbils for quantitative proteomic analyses using isobaric tags for relative and absolute quantitation (iTRAQ). Quantitative proteomic analysis of samples from two biological replicate experiments quantified a total of 2764 proteins, 166 of which were significantly altered in abundance by H. pylori infection. Pathway mapping identified significantly altered inflammatory and cancer-signaling pathways that included Rab/Ras signaling proteins. Consistent with the iTRAQ results, RABEP2 and G3BP2 were significantly up-regulated in vitro, ex vivo in primary human gastric monolayers, and in vivo in gerbil gastric epithelium following infection with H. pylori strain 7.13 in a cag-dependent manner. Within human stomachs, RABEP2 and G3BP2 expression in gastric epithelium increased in parallel with the severity of premalignant and malignant lesions and was significantly elevated in intestinal metaplasia and dysplasia, as well as gastric adenocarcinoma, compared with gastritis alone. These results indicate that carcinogenic strains of H. pylori induce dramatic and specific changes within the gastric proteome in vivo and that a subset of altered proteins within pathways with oncogenic potential may facilitate the progression of gastric carcinogenesis in humans. Gastric adenocarcinoma is the third leading cause of cancer-related death worldwide and accounts for greater than 720,000 deaths annually (1Bray F. Ren J.S. Masuyer E. Ferlay J. Global estimates of cancer prevalence for 27 sites in the adult population in 2008.Int. J. Cancer. 2013; 132: 1133-1145Crossref PubMed Scopus (1410) Google Scholar). The strongest known risk factor for this malignancy is chronic gastritis induced by the microbial pathogen Helicobacter pylori (2Polk D.B. Peek Jr., R.M. Helicobacter pylori: gastric cancer and beyond.Nat. Rev. Cancer. 2010; 10: 403-414Crossref PubMed Scopus (740) Google Scholar). H. pylori colonizes greater than 50% of the world's population (3Hooi J.K.Y. Lai W.Y. Ng W.K. Suen M.M.Y. Underwood F.E. Tanyingoh D. Malfertheiner P. Graham D.Y. Wong V.W.S. Wu J.C.Y. Chan F.K.L. Sung J.J.Y. Kaplan G.G. Ng S.C. Global Prevalence of Helicobacter pylori infection: systematic review and meta-analysis.Gastroenterology. 2017; 153: 420-429Abstract Full Text Full Text PDF PubMed Scopus (1330) Google Scholar); however, only a fraction of infected individuals ever develop cancer. The specific mechanisms by which H. pylori initiates gastric carcinogenesis are not completely understood, but disease outcomes are mediated through complex interactions between H. pylori strain-specific virulence determinants and host cell signaling responses. Initial interactions between H. pylori and the host originate at the gastric epithelial cell interface, and contact between H. pylori and gastric epithelial cells activates signaling pathways that drive oncogenesis. One microbial constituent that increases the risk for gastric cancer is the cag pathogenicity island, which encodes a bacterial type IV secretion system (T4SS) 1The abbreviations used are:cag T4SScag type IV secretion system2D LC-MS/MS2-dimensional liquid chromatography-coupled tandem mass spectrometryACNacetonitrileANOVAanalysis of varianceBHBenjamini-HochbergBLASTBasic Local Alignment Search ToolcagAcytotoxin associated gene product AcagT4SS oncogenic effector proteincagEcytotoxin associated gene product Ecag T4SSATPaseCFUcolony-forming unitsCO2carbon dioxideFBSfetal bovine serumFCSfetal calf serumFDRfalse-discovery rateG3BP2Ras GTPase-activating protein-binding protein 2GAPDHglyceraldehyde-3-phosphate dehydrogenaseGCgastric cancerH&Ehematoxylin and eosinHCDhigher energy collisional disassociationHRPhorseradish peroxidaseIACUCInstitutional Animal Care and Use CommitteeIHCimmunohistochemistryIL-1interleukin-1Il-6interleukin-6IL-8interleukin-8IL-17interleukin-17IMintestinal metaplasiaIPAIngenuity Pathway AnalysisIRBInstitutional Review BoardiTRAQisobaric tags for relative and absolute quantificationMAGmultifocal atrophic gastritisMAPKmitogen-activated protein kinaseMIFmacrophage migratory inhibitory factorMMTSmethyl methanethiosulfonateMOImultiplicity of infectionMTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide)NaClsodium chlorideNAGnonatrophic gastritisNCBINational Center for Biotechnology InformationPI3Kphosphoinositide 3-kinasePBSphosphate-buffered salineRABEP2Rab GTPase-binding effector protein 2SPEMspasmolytic-expressing metaplasiaTCEPtris (2-carboxyethyl) phosphineTEABtriethylammonium bicarbonateTh17T helper 17TNFtumor necrosis factorTNMtumor, node, and metastasis stagingTSAtrypticase soy agarUIuninfected. 1The abbreviations used are:cag T4SScag type IV secretion system2D LC-MS/MS2-dimensional liquid chromatography-coupled tandem mass spectrometryACNacetonitrileANOVAanalysis of varianceBHBenjamini-HochbergBLASTBasic Local Alignment Search ToolcagAcytotoxin associated gene product AcagT4SS oncogenic effector proteincagEcytotoxin associated gene product Ecag T4SSATPaseCFUcolony-forming unitsCO2carbon dioxideFBSfetal bovine serumFCSfetal calf serumFDRfalse-discovery rateG3BP2Ras GTPase-activating protein-binding protein 2GAPDHglyceraldehyde-3-phosphate dehydrogenaseGCgastric cancerH&Ehematoxylin and eosinHCDhigher energy collisional disassociationHRPhorseradish peroxidaseIACUCInstitutional Animal Care and Use CommitteeIHCimmunohistochemistryIL-1interleukin-1Il-6interleukin-6IL-8interleukin-8IL-17interleukin-17IMintestinal metaplasiaIPAIngenuity Pathway AnalysisIRBInstitutional Review BoardiTRAQisobaric tags for relative and absolute quantificationMAGmultifocal atrophic gastritisMAPKmitogen-activated protein kinaseMIFmacrophage migratory inhibitory factorMMTSmethyl methanethiosulfonateMOImultiplicity of infectionMTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide)NaClsodium chlorideNAGnonatrophic gastritisNCBINational Center for Biotechnology InformationPI3Kphosphoinositide 3-kinasePBSphosphate-buffered salineRABEP2Rab GTPase-binding effector protein 2SPEMspasmolytic-expressing metaplasiaTCEPtris (2-carboxyethyl) phosphineTEABtriethylammonium bicarbonateTh17T helper 17TNFtumor necrosis factorTNMtumor, node, and metastasis stagingTSAtrypticase soy agarUIuninfected. that translocates the effector protein, CagA, into host gastric epithelial cells. Intracellular CagA can become phosphorylated (4Stein M. Bagnoli F. Halenbeck R. Rappuoli R. Fantl W.J. Covacci A. c-Src/Lyn kinases activate Helicobacter pylori CagA through tyrosine phosphorylation of the EPIYA motifs.Mol. Microbiol. 2002; 43: 971-980Crossref PubMed Scopus (367) Google Scholar, 5Tammer I. Brandt S. Hartig R. Konig W. Backert S. Activation of Abl by Helicobacter pylori: a novel kinase for CagA and crucial mediator of host cell scattering.Gastroenterology. 2007; 132: 1309-1319Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 6Mueller D. Tegtmeyer N. Brandt S. Yamaoka Y. De Poire E. Sgouras D. Wessler S. Torres J. Smolka A. Backert S. c-Src and c-Abl kinases control hierarchic phosphorylation and function of the CagA effector protein in Western and East Asian Helicobacter pylori strains.J. Clin. Invest. 2012; 122: 1553-1566Crossref PubMed Scopus (173) Google Scholar) or remain unphosphorylated. In either form, CagA affects multiple host pathways that lead to alterations in cell morphology, signaling pathways, and inflammatory responses (7Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Altered states: involvement of phosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 14559-14564Crossref PubMed Scopus (668) Google Scholar, 8Odenbreit S. Puls J. Sedlmaier B. Gerland E. Fischer W. Haas R. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion.Science. 2000; 287: 1497-1500Crossref PubMed Scopus (1070) Google Scholar, 9Mimuro H. Suzuki T. Tanaka J. Asahi M. Haas R. Sasakawa C. Grb2 is a key mediator of Helicobacter pylori CagA protein activities.Mol. Cell. 2002; 10: 745-755Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 10Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA.Science. 2003; 300: 1430-1434Crossref PubMed Scopus (616) Google Scholar, 11Saadat I. Higashi H. Obuse C. Umeda M. Murata-Kamiya N. Saito Y. Lu H. Ohnishi N. Azuma T. Suzuki A. Ohno S. Hatakeyama M. Helicobacter pylori CagA targets PAR1/MARK kinase to disrupt epithelial cell polarity.Nature. 2007; 447: 330-333Crossref PubMed Scopus (379) Google Scholar). Transgenic mice that overexpress CagA develop gastric epithelial cell hyperproliferation and gastric adenocarcinoma (12Ohnishi N. Yuasa H. Tanaka S. Sawa H. Miura M. Matsui A. Higashi H. Musashi M. Iwabuchi K. Suzuki M. Yamada G. Azuma T. Hatakeyama M. Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1003-1008Crossref PubMed Scopus (455) Google Scholar), further implicating CagA as a bacterial oncoprotein. cag type IV secretion system 2-dimensional liquid chromatography-coupled tandem mass spectrometry acetonitrile analysis of variance Benjamini-Hochberg Basic Local Alignment Search Tool cytotoxin associated gene product A T4SS oncogenic effector protein cytotoxin associated gene product E ATPase colony-forming units carbon dioxide fetal bovine serum fetal calf serum false-discovery rate Ras GTPase-activating protein-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase gastric cancer hematoxylin and eosin higher energy collisional disassociation horseradish peroxidase Institutional Animal Care and Use Committee immunohistochemistry interleukin-1 interleukin-6 interleukin-8 interleukin-17 intestinal metaplasia Ingenuity Pathway Analysis Institutional Review Board isobaric tags for relative and absolute quantification multifocal atrophic gastritis mitogen-activated protein kinase macrophage migratory inhibitory factor methyl methanethiosulfonate multiplicity of infection (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide) sodium chloride nonatrophic gastritis National Center for Biotechnology Information phosphoinositide 3-kinase phosphate-buffered saline Rab GTPase-binding effector protein 2 spasmolytic-expressing metaplasia tris (2-carboxyethyl) phosphine triethylammonium bicarbonate T helper 17 tumor necrosis factor tumor, node, and metastasis staging trypticase soy agar uninfected. cag type IV secretion system 2-dimensional liquid chromatography-coupled tandem mass spectrometry acetonitrile analysis of variance Benjamini-Hochberg Basic Local Alignment Search Tool cytotoxin associated gene product A T4SS oncogenic effector protein cytotoxin associated gene product E ATPase colony-forming units carbon dioxide fetal bovine serum fetal calf serum false-discovery rate Ras GTPase-activating protein-binding protein 2 glyceraldehyde-3-phosphate dehydrogenase gastric cancer hematoxylin and eosin higher energy collisional disassociation horseradish peroxidase Institutional Animal Care and Use Committee immunohistochemistry interleukin-1 interleukin-6 interleukin-8 interleukin-17 intestinal metaplasia Ingenuity Pathway Analysis Institutional Review Board isobaric tags for relative and absolute quantification multifocal atrophic gastritis mitogen-activated protein kinase macrophage migratory inhibitory factor methyl methanethiosulfonate multiplicity of infection (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrasodium bromide) sodium chloride nonatrophic gastritis National Center for Biotechnology Information phosphoinositide 3-kinase phosphate-buffered saline Rab GTPase-binding effector protein 2 spasmolytic-expressing metaplasia tris (2-carboxyethyl) phosphine triethylammonium bicarbonate T helper 17 tumor necrosis factor tumor, node, and metastasis staging trypticase soy agar uninfected. We previously demonstrated that infection of Mongolian gerbils with a carcinogenic cag+ H. pylori strain, 7.13, rapidly induces gastric inflammation and carcinogenesis (13Franco A.T. Johnston E. Krishna U. Yamaoka Y. Israel D.A. Nagy T.A. Wroblewski L.E. Piazuelo M.B. Correa P. Peek Jr., R.M. Regulation of gastric carcinogenesis by Helicobacter pylori virulence factors.Cancer Res. 2008; 68: 379-387Crossref PubMed Scopus (228) Google Scholar, 14Noto J.M. Gaddy J.A. Lee J.Y. Piazuelo M.B. Friedman D.B. Colvin D.C. Romero-Gallo J. Suarez G. Loh J. Slaughter J.C. Tan S. Morgan D.R. Wilson K.T. Bravo L.E. Correa P. Cover T.L. Amieva M.R. Peek Jr., R.M. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.J. Clin. Invest. 2013; 123: 479-492Crossref PubMed Scopus (132) Google Scholar). Intestinal-type gastric cancer in humans progresses through a series of well-defined pathological stages from normal gastric mucosa to superficial nonatrophic gastritis, to premalignant lesions including atrophic gastritis, spasmolytic-expressing metaplasia (SPEM), intestinal metaplasia, dysplasia, and finally gastric adenocarcinoma (15Correa P. Chen V.W. Gastric cancer.Cancer Surv. 1994; 20: 55-76Google Scholar). Gerbils exhibit a similar stepwise progression of discrete histopathologic stages along the gastric carcinogenic cascade, including the development of SPEM (16Shimizu T. Choi E. Petersen C.P. Noto J.M. Romero-Gallo J. Piazuelo M.B. Washington M.K. Peek Jr, R.M. Goldenring J.R. Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori.J. Pathol. 2016; 239: 399-410Crossref PubMed Scopus (31) Google Scholar), dysplasia, and adenocarcinoma within the context of mucosal inflammation (13Franco A.T. Johnston E. Krishna U. Yamaoka Y. Israel D.A. Nagy T.A. Wroblewski L.E. Piazuelo M.B. Correa P. Peek Jr., R.M. Regulation of gastric carcinogenesis by Helicobacter pylori virulence factors.Cancer Res. 2008; 68: 379-387Crossref PubMed Scopus (228) Google Scholar, 14Noto J.M. Gaddy J.A. Lee J.Y. Piazuelo M.B. Friedman D.B. Colvin D.C. Romero-Gallo J. Suarez G. Loh J. Slaughter J.C. Tan S. Morgan D.R. Wilson K.T. Bravo L.E. Correa P. Cover T.L. Amieva M.R. Peek Jr., R.M. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.J. Clin. Invest. 2013; 123: 479-492Crossref PubMed Scopus (132) Google Scholar). Defining the mechanisms by which H. pylori initiates this cascade has important clinical implications for disease prevention and novel therapeutic interventions. Thus, we hypothesized that H. pylori induces gastric cell-specific proteomic changes critical for initiation of gastric carcinogenesis in the Mongolian gerbil model which are also important in gastric cancer progression in humans. To test this hypothesis, gastric tissue cell scrapings were harvested from uninfected gerbils and gerbils infected with the carcinogenic cag+ H. pylori strain, 7.13. Samples were isolated from gerbils prior to the development of any premalignant lesions to directly assess early proteomic changes that may initiate and drive progression along the entire gastric carcinogenic cascade. Gastric samples from two biological replicate experiments were subjected to quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). Significant H. pylori-induced proteomic alterations were analyzed relative to uninfected controls and subjected to canonical signaling and disease pathway mapping using Ingenuity Pathway Analysis (IPA) to identify critical signaling pathways altered during the early stages of H. pylori infection. Proteomic changes were then validated in in vitro and ex vivo human gastric epithelial cell-H. pylori cocultures, in vivo gerbil gastric epithelium, and in vivo human gastric tissue sections. Outbred male Mongolian gerbils were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Vanderbilt University Animal Care Facilities. Wild-type carcinogenic cag+ H. pylori strain 7.13 was minimally passaged on trypticase soy agar plates with 5% sheep blood (BD Biosciences, San Jose, CA) and in Brucella broth (BD Biosciences) supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA) for sixteen hours at 37 °C with 5% CO2. Gerbils were orogastrically challenged with sterile Brucella broth (negative control), wild-type cag+ H. pylori strain 7.13, or a cagE− isogenic mutant, and gerbils were euthanized 6 weeks post-challenge, as previously described (14Noto J.M. Gaddy J.A. Lee J.Y. Piazuelo M.B. Friedman D.B. Colvin D.C. Romero-Gallo J. Suarez G. Loh J. Slaughter J.C. Tan S. Morgan D.R. Wilson K.T. Bravo L.E. Correa P. Cover T.L. Amieva M.R. Peek Jr., R.M. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.J. Clin. Invest. 2013; 123: 479-492Crossref PubMed Scopus (132) Google Scholar). The Vanderbilt University Institutional Animal Care and Use Committee (IACUC) approved all experiments and procedures. Linear strips of gastric tissue, extending from the squamocolumnar junction through the proximal duodenum were harvested and homogenized in sterile phosphate-buffered saline (PBS, Corning, Corning, NY). Following serial dilution, samples were plated on selective trypticase soy agar (TSA, Remel) plates with 5% sheep blood (Hemostat Laboratories, Dixon, CA) containing vancomycin (Sigma-Aldrich, St. Louis, MO, 20 μg/ml), nalidixic acid (Sigma-Aldrich, 10 μg/ml), bacitracin (Calbiochem, San Diego, CA, 30 μg/ml), and amphotericin B (Sigma-Aldrich, 2 μg/ml) for selection, isolation and quantification of H. pylori, as previously described (14Noto J.M. Gaddy J.A. Lee J.Y. Piazuelo M.B. Friedman D.B. Colvin D.C. Romero-Gallo J. Suarez G. Loh J. Slaughter J.C. Tan S. Morgan D.R. Wilson K.T. Bravo L.E. Correa P. Cover T.L. Amieva M.R. Peek Jr., R.M. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.J. Clin. Invest. 2013; 123: 479-492Crossref PubMed Scopus (132) Google Scholar). Plates were incubated for 3 to 5 days at 37 °C with 5% CO2. Colonies were identified as H. pylori based on characteristic spiral morphology, Gram stain (Becton, Dickinson and Company, Franklin Lakes, NJ), and urease and oxidase enzyme activities (Becton, Dickinson and Company). Colony counts were expressed as log colony-forming units (CFU) per gram of gastric tissue. Linear strips of gastric tissue, extending from the squamocolumnar junction through the proximal duodenum, were fixed in 10% neutral-buffered formalin (Azer Scientific, Morgantown, PA), paraffin-embedded, and stained with hematoxylin and eosin (H&E) as well as with a modified Steiner stain for detection of H. pylori. A single pathologist (MBP), blinded to treatment groups, assessed and scored indices of inflammation and injury and topography of colonization by Steiner stain. Severity of acute and chronic inflammation was graded on a scale from 0–3 (absent (0), mild (1), moderate (2), or marked inflammation (3)) in both the gastric antrum and corpus, leading to a maximum cumulative score of twelve, as previously described (14Noto J.M. Gaddy J.A. Lee J.Y. Piazuelo M.B. Friedman D.B. Colvin D.C. Romero-Gallo J. Suarez G. Loh J. Slaughter J.C. Tan S. Morgan D.R. Wilson K.T. Bravo L.E. Correa P. Cover T.L. Amieva M.R. Peek Jr., R.M. Iron deficiency accelerates Helicobacter pylori-induced carcinogenesis in rodents and humans.J. Clin. Invest. 2013; 123: 479-492Crossref PubMed Scopus (132) Google Scholar). Gastric cell scrapings were harvested from linear strips of gerbil gastric tissue, extending from the squamocolumnar junction through the proximal duodenum, and were solubilized in RIPA buffer (50 mm Tris, pH 7.2; 150 mm NaCl; 1% Triton X-100; and 0.1% SDS). Gastric cell scrapings were used to examine proteins from the heterogeneous cell populations of the gastric mucosa. Gastric proteins samples from H. pylori-infected tissues (n = 3) were pooled together, as were gastric proteins samples from uninfected tissue (n = 3) for each independent replicate experiment to achieve statistical power to detect significant differences, as previously performed (17Noto J.M. Chopra A. Loh J.T. Romero-Gallo J. Piazuelo M.B. Watson M. Leary S. Beckett A.C. Wilson K.T. Cover T.L. Mallal S. Israel D.A. Peek R.M. Pan-genomic analyses identify key Helicobacter pylori pathogenic loci modified by carcinogenic host microenvironments.Gut. 2017; 67: 1793-1804Crossref PubMed Scopus (20) Google Scholar). An equivalent amount of protein was taken from each biological sample, such that the pooled sample contained 100 μg of protein in total. Gastric protein samples were precipitated with ice-cold acetone overnight at −20 °C. Following precipitation, samples were centrifuged at 18,000 × g at 4 °C, and precipitates were washed with cold acetone, dried, and reconstituted in 8 m urea in 250 mm triethylammonium bicarbonate buffer (TEAB, pH 8.0). Samples were reduced with 5 μl of 50 mm tris (2-carboxyethyl) phosphine (TCEP), alkylated with 2.5 μl of 200 mm methyl methanethiosulfonate (MMTS), diluted with TEAB to obtain a final solution containing 2 m urea, and digested with sequencing-grade trypsin (Promega, Madison, WI) overnight. To facilitate quantitative analysis, peptides were labeled with iTRAQ reagents (AB Sciex, Concord, Ontario, Canada), according to the manufacturer's instructions. For each 50 μg of protein, one unit of labeling reagent was used. Labeling reagent was reconstituted in ethanol, such that each protein sample was labeled at a final concentration of 90% ethanol, and labeling was performed for two hours. Pooled lysates from H. pylori-infected or uninfected gastric tissue were labeled with 4-plex iTRAQ reagent 117 or 115, respectively. Two-plex iTRAQ comparisons were then conducted. The resulting labeled peptides were desalted by a modified Stage-tip method, as previously described (18Voss B.J. Loh J.T. Hill S. Rose K.L. McDonald W.H. Cover T.L. Alteration of the Helicobacter pylori membrane proteome in response to changes in environmental salt concentration.Proteomics Clin. Appl. 2015; 9: 1021-1034Crossref PubMed Scopus (29) Google Scholar). iTRAQ-labeled samples were mixed and acidified with trifluoroacetic acid (TFA). A disc of C18 extraction membrane (C18 SPE Empore disk, Chrom Tech Inc., Apple Valley, MN) was cored with a 16-gauge needle, and the cored piece of membrane was fitted tightly into a 200 μl pipette tip. Three mg of C18 resin (Jupiter C18, 5 μm particle size, Phenomenex, Torrance, CA) were suspended in 200 μl of methanol and loaded into the pipette tip containing the cored C18 membrane. The C18 material was packed into the tip using centrifugation to form a resin-packed C18 clean-up tip (resin tip). Resin tips were equilibrated with 0.1% TFA in HPLC-grade water (Fisher Scientific, Waltham, MA), labeled peptides were loaded into the tip by centrifugation, washed with 0.1% TFA, and eluted with 100 μl of 80% acetonitrile (ACN) containing 0.1% TFA. Eluted peptides were dried by speed vacuum centrifugation, and then peptides were reconstituted in 0.1% formic acid and analyzed by 2-dimensional liquid chromatography-coupled tandem mass spectrometry (2D LC-MS/MS). Peptides were loaded onto a self-packed biphasic C18/SCX MudPIT column using a Helium-pressurized cell (pressure bomb). The MudPIT column consisted of 360 × 150 μm i.d. fused silica, which was fitted with a filter-end fitting (IDEX Health & Science, Oak Harbor, WA) and packed with 6 cm of Luna SCX material (5 μm, 100Å) followed by 4 cm of Jupiter C18 material (5 μm, 300Å, Phenomenex). Once samples were loaded, the MudPIT column was connected using an M-520 microfilter union (IDEX Health & Science) to an analytical column (360 μm × 100 μm i.d.), equipped with a laser-pulled emitter tip and packed with 20 cm of C18 reverse phase material (Jupiter, 3 μm beads, 300Å, Phenomenex). Using a Dionex Ultimate 3000 nanoLC and autosampler, MudPIT analysis was performed with 13 salt steps (0, 25, 50, 75, 100, 150, 200, 250, 300, 500, 1 m, 2 m, and 5 m ammonium acetate). Following each salt pulse delivered by the autosampler, peptides were gradient-eluted from the reverse analytical column at a flow rate of 350 nL/min. Mobile phase solvents (HPLC-grade) consisted of 0.1% formic acid, 99.9% water (solvent A) and 0.1% formic acid, 99.9% acetonitrile (solvent B). For the peptides from the first 11 SCX fractions, the reverse phase gradient consisted of 2–50% B in 80 min, followed by a 10-min equilibration at 2% solvent B. For the last 2 SCX-eluted peptide fractions, the peptides were eluted from the reverse phase analytical column using a gradient of 2–98% solvent B in 80 min, followed by a 10-min equilibration at 2% solvent B. A Q Exactive Plus mass spectrometer (Thermo Scientific, Waltham, WA), equipped with a nanoelectrospray ionization source, was used to mass analyze the eluting peptides. The Q Exactive instrument was operated in data-dependent mode acquiring higher energy collisional disassociation (HCD) MS/MS scans (r = 17,500) after each MS1 scan on the 20 most abundant ions using an MS1 ion target of 3 × 106 ions and an MS2 target of 1 × 105 ions. The HCD-normalized collision energy was set to 30, dynamic exclusion was set to 30 s, and peptide match and isotope exclusion were enabled. Mass spectra were processed using the Spectrum Mill software package (version B.04.00, Agilent Technologies) and were searched against a database containing the Mus musculus subset of the UniprotKB protein database (www.uniprot.org, UniProtKB Release 2012_06, 16,651 protein entries). MS/MS spectra acquired on the same precursor m/z (±0.01 m/z) within ±1 s in retention were merged. MS/MS spectra of poor quality, which did not have a sequence tag length >1, were excluded. A minimum matched peak intensity requirement was set to 50%. Additional search parameters included: trypsin enzyme specificity with a maximum of three missed cleavages, ±20 ppm precursor mass tolerance, ±20 ppm (HCD) product mass tolerance, and fixed modifications including MMTS alkylation of cysteines and iTRAQ labeling of lysines and peptide N termini. Oxidation of methionine was allowed as a variable modification. Autovalidation was performed such that peptide assignments to mass spectra were designated as valid following an automated procedure during which score thresholds were optimized separately for each precursor charge state, and the maximum target-decoy-based false-discovery rate (FDR) was set to 1.0% (19Mertins P. Udeshi N.D. Clauser K.R. Mani D.R. Patel J. Ong S.E. Jaffe J.D. Carr S.A. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics.Mol. Cell. Proteomics. 2012; 11 (M111.014423)Abstract Full Text Full Text PDF Scopus

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