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Comparative Proteomics Analysis of Barrett Metaplasia and Esophageal Adenocarcinoma Using Two-dimensional Liquid Mass Mapping*S

  • Jia Zhao
    Affiliations
    Department of Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Andrew C. Chang
    Affiliations
    Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Chen Li
    Affiliations
    Department of Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Kerby A. Shedden
    Affiliations
    Department of Statistics, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Dafydd G. Thomas
    Affiliations
    Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • David E. Misek
    Affiliations
    Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Arun Prasad Manoharan
    Affiliations
    Department of Bioinformatics, University of Michigan and Departments, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Thomas J. Giordano
    Affiliations
    Department of Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Pathology, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • David G. Beer
    Affiliations
    Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • David M. Lubman
    Correspondence
    To whom correspondence should be addressed: Dept. of Surgery, University of Michigan, Ann Arbor, MI 48109. Tel.: 734-647-8834; Fax: 734-615-2088
    Affiliations
    Department of Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109

    Department of Comprehensive Cancer Center, University of Michigan Medical Center, Ann Arbor, Michigan 48109
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  • Author Footnotes
    * This work was supported in part by the NCI, National Institutes of Health Grant CA71606 (to D. G. B.) and National Institutes of Health Grants R01CA10010 (to D. M. L.), R01CA90503 (to D. M. L.), and R01GM49500 (to D. M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
Open AccessPublished:July 08, 2006DOI:https://doi.org/10.1074/mcp.M600175-MCP200
      Esophageal adenocarcinoma, currently the seventh leading cause of cancer-related death, has been associated with the presence of Barrett metaplasia. The malignant potential of Barrett metaplasia is evidenced by ultimate progression of this condition to invasive adenocarcinoma. We utilized liquid phase separation of proteins with chromatofocusing in the first dimension and nonporous reverse phase HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify proteins differentially expressed in six Barrett metaplasia samples as compared with six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue. Approximately 300 protein bands were detected by mass mappings, and 38 differentially expressed proteins were identified by μLC-MS/MS. The false positive rates of the peptide identifications were evaluated by reversed database searching. Among the proteins that were identified, Rho GDP dissociation inhibitor 2, α-enolase, Lamin A/C, and nucleoside-diphosphate kinase A were demonstrated to be up-regulated in both mRNA and protein expression in esophageal adenocarcinomas relative to Barrett metaplasia. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays. The cellular expression patterns were verified in both esophageal adenocarcinomas and in Barrett metaplasia by immunohistochemistry. These differentially expressed proteins may have utility as useful candidate markers of esophageal adenocarcinoma.
      Esophageal adenocarcinoma is increasing rapidly in Western countries and is currently the seventh leading cause of cancer-related death (
      • Jemal A.
      • Thomas A.
      • Murray T.
      • Thun M.
      Cancer statistics, 2002.
      ). Esophageal adenocarcinoma has been associated with the presence of Barrett metaplasia, a condition in which the normal squamous epithelium of the esophagus is replaced by columnar epithelium. The malignant potential of this condition is evidenced by the progression of Barrett metaplasia to low grade dysplasia, high grade dysplasia, and ultimately to invasive adenocarcinoma. The risk of developing adenocarcinoma is 30–125 times higher in people who have Barrett metaplasia than people who do not. The prognosis of patients with esophageal adenocarcinoma remains poor with overall 5-year survival rates of only 5–15% (
      • Jemal A.
      • Thomas A.
      • Murray T.
      • Thun M.
      Cancer statistics, 2002.
      ). Unfortunately patients often present with regionally advanced disease (
      • Jemal A.
      • Siegel R.
      • Ward E.
      • Murray T.
      • Xu J.
      • Smigal C.
      • Thun M.J.
      Cancer statistics, 2006.
      ). Given the poor prognosis associated with esophageal adenocarcinoma, it is imperative to improve our understanding of the tumorigenesis and the factors associated with increased risk. It is possible that therapeutic targets or protein markers can be identified that will ultimately facilitate improved patient survival.
      Proteomics technologies have been used for the identification of candidate markers for early cancer detection (
      • Tyers M.
      • Mann M.
      From genomics to proteomics.
      ). The global analysis of protein expression complements genomics analyses. For example, proteomics analysis may provide further insight into post-translational modifications affecting cellular function that otherwise could not be identified by genomics analysis. It is important to identify changes in global protein expression to identify specific proteins that are involved in cancer-related processes. We and others have demonstrated that two-dimensional (2-D)
      The abbreviations used are: 2-D, two-dimensional; NPS-RP, nonporous reverse phase; CF, chromatofocusing; OG, n-octyl β-d-glucopyranoside; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RhoGDI, Rho GDP dissociation inhibitor.
      1The abbreviations used are: 2-D, two-dimensional; NPS-RP, nonporous reverse phase; CF, chromatofocusing; OG, n-octyl β-d-glucopyranoside; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; RhoGDI, Rho GDP dissociation inhibitor.
      liquid mass mapping can be used for quantitative and comparative proteomics analyses (
      • Lubman D.M.
      • Kachman M.T.
      • Wang H.
      • Gong S.
      • Yan F.
      • Hamler R.L.
      • O'Neil K.A.
      • Zhu K.
      • Buchanan N.S.
      • Barder T.J.
      Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates.
      ,
      • Hamler R.L.
      • Zhu K.
      • Buchanan N.S.
      • Kreunin P.
      • Kachman M.T.
      • Miller F.R.
      • Lubman D.M.
      A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification.
      ,
      • Zhao J.
      • Zhu K.
      • Lubman D.M.
      • Miller F.R.
      • Shekhar M.P.
      • Gerard B.
      • Barder T.J.
      Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique.
      ). Ion intensity-based quantitative approaches have progressively gained more popularity as mass spectrometry performance has improved significantly. 2-D fractionation techniques before mass detection simplify the complex proteome. Because mass is a unique tag for intact proteins, this method avoids the problem in quantitation induced by incomplete separation. In addition, liquid phase analysis allows easy interface to mass spectrometry analysis.
      In this study, we evaluated protein expression differences to identify markers of disease progression of Barrett metaplasia to esophageal cancer to gain further insight into potential mechanisms underlying these changes. Protein lysates were prepared from both high grade dysplasia and esophageal adenocarcinoma tissue, both obtained from the same six patients. These lysates were resolved by 2-D LC using chromatofocusing in the first dimension and nonporous silica reverse phase (NPS-RP) HPLC in the second dimension. Separated proteins in liquid phase were ionized by ESI-TOF, and the intact molecular weights were obtained after deconvolution of multiple charged peaks. Proteins were quantified based on individual molecular weight intensity and assembly of a protein mass map. Hierarchical clustering of the mass maps correctly segregated each of the 12 samples by histological type into Barrett metaplasia or esophageal adenocarcinoma. Differentially expressed proteins were identified by μLC-MS/MS after protease digestion. Candidate proteins were examined at the mRNA level using high density oligonucleotide microarrays as well as at the protein level using immunohistochemistry to verify cellular expression patterns. The 2-D mass maps and the corresponding protein identification have utility for analysis of cellular protein expression changes that are associated with progression of Barrett metaplasia to esophageal adenocarcinoma.

      DISCUSSION

      We utilized liquid phase separation of proteins incorporating chromatofocusing in the first dimension and NPS-RP HPLC in the second dimension followed by ESI-TOF mass spectrometry to identify multiple proteins differentially expressed in the progression from Barrett metaplasia to esophageal adenocarcinoma. In our analysis, we used six Barrett metaplasia samples and six esophageal adenocarcinoma samples; all six Barrett samples were obtained from the identical six patients from whom we obtained the esophageal adenocarcinoma tissue, thereby eliminating possible genetic polymorphisms between preneoplastic and neoplastic tissues as a confounding variable. This increased expression was verified at the messenger level and further confirmed by immunohistochemistry for several of these proteins, notably Rho GDP dissociation inhibitor 2 and α-enolase. This approach of candidate identification with subsequent verification has strong potential for identifying candidate protein markers and mechanisms involved in the tumorigenesis of esophageal adenocarcinoma. In contrast to large scale analysis of transcriptional regulation, this combined approach has the additional capability to identify post-translationally modified proteins. We identified several such candidates, including Calgranulin B, whose mRNA levels did not differ significantly in adenocarcinoma compared with Barrett metaplasia samples.
      Furthermore with our clustering analysis we observed that both metaplastic and neoplastic tissues segregated into two large groups, recapitulating their designated histology. This is reflective of the differential protein expression between Barrett metaplasia and esophageal adenocarcinoma that we demonstrated for several individual proteins. Although our findings require extensive validation in larger independent sets of tissue samples, this high throughput approach may have broad potential for tumor identification and classification as we have demonstrated previously in other tissue types (
      • Wang Y.
      • Wu R.
      • Cho K.R.
      • Shedden K.A.
      • Barder T.J.
      • Lubman D.M.
      Classification of cancer cell lines using an automated two-dimensional liquid mapping method with hierarchical clustering techniques.
      ) and as others have suggested for a variety of proteomics strategies (
      • Alaiya A.A.
      • Franzen B.
      • Hagman A.
      • Dysvik B.
      • Roblick U.J.
      • Becker S.
      • Moberger B.
      • Auer G.
      • Linder S.
      Molecular classification of borderline ovarian tumors using hierarchical cluster analysis of protein expression profiles.
      ,
      • Yanagisawa K.
      • Shyr Y.
      • Xu B.J.
      • Massion P.P.
      • Larsen P.H.
      • White B.C.
      • Roberts J.R.
      • Edgerton M.
      • Gonzalez A.
      • Nadaf S.
      • Moore J.H.
      • Caprioli R.M.
      • Carbone D.P.
      Proteomic patterns of tumour subsets in non-small-cell lung cancer.
      ).
      Currently detection of esophageal cancer, especially adenocarcinoma arising in Barrett esophagus, relies on histological examination of multiple endoscopic biopsies obtained at regularly spaced intervals in a segment of abnormal-appearing esophagus. Given the poor prognosis associated with this disease, a better understanding of the pathogenesis of the disease is essential. Utilization of 2-D mass maps with corresponding protein identification can provide detailed analyses of cellular protein expression changes that are associated with esophageal adenocarcinoma progression. These identified proteins may have utility as candidate markers of progression from Barrett metaplasia to esophageal adenocarcinoma.

      Acknowledgments

      We thank Alexey Nesvizhskii for help on the Protein/PeptideProphet software.

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