An approach to proteomic analysis of human tumors†
Michael R. Emmert-Buck
Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland
Cancer Genome Anatomy Project, Office of the Director, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorJohn W. Gillespie
Cancer Genome Anatomy Project, Office of the Director, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorCloud P. Paweletz
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, Maryland
Department of Chemistry, Georgetown University, Washington, D.C.
Search for more papers by this authorDavid K. Ornstein
Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland
Urologic Oncology Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorVenkatesha Basrur
Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorEttore Appella
Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorQuan-Hong Wang
Pathology Laboratory, Shanxi Cancer Hospital, Taiyuan, China
Search for more papers by this authorJing Huang
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorNan Hu
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorPhil Taylor
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorCorresponding Author
Emanuel F. Petricoin III
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, Maryland
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, FDASearch for more papers by this authorMichael R. Emmert-Buck
Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland
Cancer Genome Anatomy Project, Office of the Director, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorJohn W. Gillespie
Cancer Genome Anatomy Project, Office of the Director, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorCloud P. Paweletz
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, Maryland
Department of Chemistry, Georgetown University, Washington, D.C.
Search for more papers by this authorDavid K. Ornstein
Pathogenetics Unit, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland
Urologic Oncology Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorVenkatesha Basrur
Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorEttore Appella
Laboratory of Cell Biology, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorQuan-Hong Wang
Pathology Laboratory, Shanxi Cancer Hospital, Taiyuan, China
Search for more papers by this authorJing Huang
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorNan Hu
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorPhil Taylor
Cancer Prevention Studies Branch, National Cancer Institute, Bethesda, Maryland
Search for more papers by this authorCorresponding Author
Emanuel F. Petricoin III
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, Maryland
Tissue Proteomics Unit, Division of Cytokine Biology, Center for Biologics Evaluation Research, FDASearch for more papers by this authorThis article is a US Government work and, as such, is in the public domain in the United States of America.
Abstract
A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3–10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol. Carcinog. 27:158–165, 2000. Published by Wiley-Liss Inc.
REFERENCES
- 1 Wilkins MR, Sanchez JC, Gooley AA, et al. Progress with proteome projects: Why all proteins expressed by a genome should be identified and how to do it. Biotech Gen Eng Rev 1996; 13: 19–50.
- 2 Kovarova H, Stulik J, Hochstrasser DF, Bures J, Melichar B, Jandik P. Two-dimensional electrophoretic study of normal colon mucosa and colorectal cancer. Appl Theor Electrophoresis 1994; 4: 103–106.
- 3 Laswson SR, Latter G, Miller DS, et al. Quantitative protein changes in metastatic versus primary epithelial ovarian carcinoma. Gynecol Oncol 1991; 41: 22–27.
- 4 Okuzawa K, Franzen B, Lindholm J, et al. Characterization of gene expression in clinical lung cancer materials by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 1994; 15: 382–390.
- 5 Celis JE, Ostergaard M, Basse B, et al. Loss of adipocyte-type fatty acid binding protein and other protein biomarkers is associated with progression of human bladder transitional cell carcinomas. Cancer Res 1996; 56: 4782–4790.
- 6 Giometti CS, Williams K, Tollaksen SL. A two-dimensional electrophoresis database of human breast epithelial cell proteins. Electrophoresis 1997; 16: 1187–1189.
- 7 Sarto C, Marocchi A, Sanchez JC, et al. Renal cell carcinoma and normal kidney protein expression. Electrophoresis 1997; 18: 599–604.
- 8 Reymond MA, Sanchez JC, Schneider C, et al. Specific sample preparation in colorectal cancer. Electrophoresis 1997; 18: 622–624.
- 9 Franzen B, Hirano T, Okuzawa K, et al. Sample preparation of human tumors prior to two-dimensional electrophoresis of proteins. Electrophoresis 1995; 16: 1087–1089.
- 10 Emmert-Buck MR, Roth MJ, Zhuang Z. Increased gelatinase A and cathepsin B activity in invasive tumor regions of human colon cancer samples. Am J Pathol 1994; 145: 1285–1290.
- 11 Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science 1996; 274: 998–1001.
- 12 Bonner RF, Emmert-Buck MR, Cole KA, et al. Laser capture microdissection: molecular analysis of tissue. Science 1997; 278: 1481–1483.
- 13 Matsui N, Smith DM, Clauser KR, et al. Immobilized pH gradient two-dimensional gel electrophoresis and mass spectrometric identification of cytokine-regulated proteins in ME-180 cervical carcinoma cells. Electrophoresis 1997; 18: 409–417.
- 14 Li G, Waltham M, Anderson NL, Unsworth E, Treston A, Weinstein JN. Rapid mass spectrometric identification of proteins from two-dimensional polyacrylamide gels after in gel proteolytic digestion. Electrophoresis 1997; 18: 391–402.
- 15 Hunt DF, Henderson RA, Shabanowitz J, et al. Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 1992; 255: 1261–1263.
- 16 Mann M, Wilm M. Error-tolerant identification of peptides in sequence databases by peptide sequence tags. Anal Chem 1994; 66: 4390–4399.
- 17 Eng J, McCormack AL, Yates JR III. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Mass Spectrom 1994; 5: 976–989.
- 18
Banks RE,
Dunn MJ,
Forbes MA, et al.
The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis—preliminary findings.
Electophoresis
1999;
20: 689–700.
10.1002/(SICI)1522-2683(19990101)20:4/5<689::AID-ELPS689>3.0.CO;2-J CAS PubMed Web of Science® Google Scholar
- 19 Krizman DB, Chuaqui RF, Meltzer PS, et al. Construction of a representative cDNA library from prostatic intraepithelial neoplasia (PIN). Cancer Res 1996; 56(23): 5380–5383.
- 20 Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 1996; 20: 2057–2059.
- 21 Chuaqui RF, Englert CR, Strup SE, et al. Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma. Urology 1997; 50(2): 302–307.
- 22 Hung J, Kishimoto Y, Sugio K, et al. Allel-specific chromosome 3p deletions occur at an early stage in the pathogenesis of lung carcinoma. JAMA 1995; 273(7): 558–563.
- 23 Luo L, Salunga RC, Guo H, et al. Gene expression profiles of laser-captured adjacent neuronal subtypes. Nat Med 1999; 5(1): 117–122.
- 24 Wiltshire RN, Duray PH, Bittner ML, et al. Direct visualization of the clonal progression of primary cutaneous melanoma: application of tissue microdissection and comparative genomic hybridization. Cancer Res 1995; 55(18): 3954–3957.
- 25 Emmert-Buck MR, Lubensky IA, Dong Q, et al. Localization of the multiple endocrine neoplasia type I (MEN1) gene based on tumor deletion mapping. Cancer Res 1997; 57(10): 1855–1858.
- 26
Moskaluk CA,
Rumpel CA.
Allelic deletion in 11p15 is a common occurrence in esophageal and gastric adenocarcinoma.
Cancer
1998;
83(2): 232–239.
10.1002/(SICI)1097-0142(19980715)83:2<232::AID-CNCR5>3.0.CO;2-S CAS PubMed Web of Science® Google Scholar
- 27 Smith KJ, Johnson KA, Bryan TM, et al. The APC gene product in normal and tumor cells. Proc Natl Acad Sci USA 1993; 90(7): 2846–2850.
- 28
Bostwick DG,
Brawer MK.
Prostatic intra-epithelial neoplasia and early invasion in prostate cancer.
Cancer
1987;
59: 788–794.
10.1002/1097-0142(19870215)59:4<788::AID-CNCR2820590421>3.0.CO;2-I CAS PubMed Web of Science® Google Scholar
- 29
Page D,
Dupont WD.
Anatomical markers of human premalignancy and risk of breast cancer.
Cancer
1990;
66: 1326–1335.
10.1002/1097-0142(19900915)66:14+<1326::AID-CNCR2820661405>3.0.CO;2-P PubMed Web of Science® Google Scholar
- 30 Sanchez JC, Wirth P, Jaccoud S, et al. Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots. Electrophoresis 1997; 18: 599–604.
- 31 Pencil SD, Toth M. Elevated levels of annexin I protein in vitro and in vivo in rat and human mammary adenocarcinoma. Clin Exp Metastasis 1998; 16(2): 113–121.
- 32 Schwartz-Albiez R, Koretz K, Moller P, Wirl G. Differential expression of annexins I and II in normal and malignant human mammary epithelial cells. Differentiation 1993; 52(3): 229–237.
- 33 Sinha P, Hutter G, Kottgen E, Dietel M, Schadendorf D, Lage H. Increased expression of annexin I and thioredoxin detected by two-dimensional gel electrophoresis of drug resistant human stomach cancer cells. J Biochem Biophys Meth 1998; 37(3): 105–161.
- 34 Ahn SH, Sawada H, Ro JY, Nicolson GL. Differential expression of annexin I in human mammary ductal epithelial cells in normal and benign and malignant breast tissues. Clin Exp Metastatis 1997; 15 (2): 151–156.
- 35 Masaki T, Tokuda M, Ohnishi M, et al. Enhanced expression of the protein kinase substrate annexin in human hepatocellular carcinoma. Hepatology 1996; 24(1): 72–81.
- 36 Grace MP, Kim KH, True LD, Fuchs E. Keratin expression in normal esophageal epithelium and squamous cell carcinoma of the esophagus. Cancer Res 1985; 45 (2): 841–846.
- 37 Debus E, Moll R, Franke WW, Weber K, Osborn M. Immunohistochemical distinction of human carcinomas by cytokeratin typing with monoclonal antibodies. Am J Pathol 1984; 14 (1): 121–130.
