2D Differential In-gel Electrophoresis for the Identification of Esophageal Scans Cell Cancer-specific Protein Markers

Abstract

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1′-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of ∼ 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers. The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1′-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of ∼ 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers. Proteomics (1Wilkins M.R. Sanchez J.C. Gooley A.A. Appel R.D. Humphery-Smith I. Hochstrasser D.F. Williams K.L. Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it.Biotechnol. Genet. Eng. Rev. 1996; 13: 19-50Google Scholar) includes the systematic cataloging of protein expression on a large scale, providing complementary information to that obtained from mRNA profiling by microarray (2Gygio S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Google Scholar, 3Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar). Such studies could lead to the molecular characterization of cellular events associated with cancer progression, cellular signaling, and developmental stages (4Lewis T.S. Hunt J.B. Aveline L.D. Jonscher K.R. Louie D.F. Yeh J.M. Nahreini T.S. Resing K.A. Ahn N.G. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry.Mol. Cell. 2000; 6: 1343-1354Google Scholar, 5Hanash S.M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status.Electrophoresis. 2000; 21: 1202-1209Google Scholar, 6Reymond M.A. Sanchez J.C. Hughes G.J. Gunther K. Riese J. Tortola S. Peinado M.A. Kirchner T. Hohenberger W. Hochstrasser D.F. Kockerling F. Standardized characterization of gene expression in human colorectal epithelium by two-dimensional electrophoresis.Electrophoresis. 1997; 18: 2842-2848Google Scholar, 7Bichsel V.E. Liotta L.A. Petricoin III, E.F. Cancer proteomics: from biomarker discovery to signal-pathway profiling.Cancer J. 2001; 7: 69-78Google Scholar). Proteomics studies of clinical tumor samples have led to the identification of cancer-specific protein markers, which provide a basis for developing new methods for early diagnosis and early detection and clues to understand the molecular characterization of cancer progression (5Hanash S.M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status.Electrophoresis. 2000; 21: 1202-1209Google Scholar, 8Prasannan L. Misek D.E. Hinderer R. Michon J. Geiger J.D. Hanash S.M. Identification of β-tubulin isoforms as tumor antigens in neuroblastoma.Clin. Cancer Res. 2000; 6: 3949-3956Google Scholar, 9Liotta L. Petricoin E. Molecular profiling of human cancer.Nat. Rev. Genet. 2000; 1: 48-56Google Scholar, 10Emmert-Buck M.R. Gillespie J.W. Paweletz C.P. Ornstein D.K. Basrur V. Appella E. Wang Q.H. Huang J. Hu N. Taylor P. Petricoin III, E.F. An approach to proteomic analysis of human tumors.Mol. Carcinog. 2000; 27: 158-165Google Scholar). A mainstay of conventional proteomics is high resolution 2D 1The abbreviations used are: 2D, two dimensional; CHAPS, 3-([(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Cy3, 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxy-succinimidyl ester; Cy5, 1-(5-carboxypentyl)-1′-methylindodi-carbocyanine halide N-hydroxysuccinimidyl ester; DIGE, differential in-gel electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; LCQ, liquid chromatography electrospray ion trap mass spectrometer; LCM, laser capture microdissection; DTT, dithiothreitol; 3D, three dimensional. 1The abbreviations used are: 2D, two dimensional; CHAPS, 3-([(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Cy3, 1-(5-carboxypentyl)-1′-propylindocarbocyanine halide N-hydroxy-succinimidyl ester; Cy5, 1-(5-carboxypentyl)-1′-methylindodi-carbocyanine halide N-hydroxysuccinimidyl ester; DIGE, differential in-gel electrophoresis; HPLC, high performance liquid chromatography; MS, mass spectrometry; LCQ, liquid chromatography electrospray ion trap mass spectrometer; LCM, laser capture microdissection; DTT, dithiothreitol; 3D, three dimensional. gel electrophoresis (11O'Farrell P.H. High resolution two-dimensional electrophoresis of proteins.J. Biol. Chem. 1975; 250: 4007-4021Google Scholar, 12Gorg A. Postel W. Gunther S. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 1988; 9: 531-546Google Scholar) followed by protein identification using mass spectrometry (13Dongre A.R. Eng J.K. Yates III, J.R. Emerging tandem-mass-spectrometry techniques for the rapid identification of proteins.Trends Biotechnol. 1997; 15: 418-425Google Scholar, 14Gygi S.P. Han D.K. Gingras A.C. Sonenberg N. Aebersold R. Protein analysis by mass spectrometry and sequence database searching: tools for cancer research in the post-genomic era.Electrophoresis. 1999; 20: 310-319Google Scholar, 15Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. The yeast nuclear pore complex: composition, architecture, and transport mechanism.J. Cell Biol. 2000; 148: 635-651Google Scholar). The state of the art 2D gel system can be loaded with a few milligrams of protein and separates thousands of protein spots (5Hanash S.M. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status.Electrophoresis. 2000; 21: 1202-1209Google Scholar, 16Gorg A. Obermaier C. Boguth G. Harder A. Scheibe B. Wildgruber R. Weiss W. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 2000; 21: 1037-1053Google Scholar). Although the technique has been widely used and successfully applied in a variety of biological systems, several technical limitations exist. Because of subtle changes in experimental conditions, the protein expression patterns on a single 2D gel usually cannot be fully duplicated, which makes it difficult to find the proteins changed between gels and to quantify the changes in protein expression. Although a comparison of protein expression profiles from regular 2D gel electrophoresis can be carried out with the assistance of various software programs, it typically requires some computerized justification of 2D gel images so that two images can be superimposed and compared. These difficulties limit the speed and accuracy of quantitation of protein spots in 2D gel electrophoresis. The differential in-gel electrophoresis (DIGE) technique recently introduced by Amersham Biosciences, Inc. is aimed at improving reproducibility. The concept of DIGE was originally developed by Minden and colleagues (17Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Google Scholar). To analyze the samples in DIGE, two pools of protein extracts are labeled covalently with fluorescent cyanine dyes, Cy3 and Cy5, respectively. These labeled proteins are mixed and separated in the same 2D gel. The 2D gel patterns can be rapidly imaged by the fluorescence excitation of either Cy3 or Cy5 dyes. The amount of the dye is controlled in such a way that on average one protein molecule is labeled not more than once, and the minimum number of the molecules of each protein are labeled. A comparison of the resulting images allows quantitation of each protein spot. Because two pools of the proteins are separated in the same gel, those proteins existing in both pools will migrate to the same locations in the 2D gel, minimizing the reproducibility problem. Quantitation of the protein profile can be rapidly and accurately achieved based on the fluorescence intensity. Recently, this technique was used by Davison and colleagues (18Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Posgnan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Google Scholar) for proteomics studies of mouse liver homogenates to examine the molecular basis of the hepatotoxin, N-acetyl-p-aminophenol. They demonstrated that the DIGE technology has adequate sensitivity and reproducibility and wide dynamic range. We have applied the DIGE technique for the identification of esophageal squamous cell cancer-specific protein markers. Both esophageal cancer and normal squamous epithelium cells were procured from the same esophageal tumor sample by laser capture microdissection (LCM). 200 μg of whole cell extracts of both pools were labeled, mixed, and separated in an 18-cm × 18-cm gel. Analysis of the produced 2D images identified 1038 protein spots in cancer cell lysate and 1088 protein spots in normal cell lysate, of which 58 protein spots were found up-regulated by >3-fold, and 107 proteins were down-regulated by >3-fold in cancer cells. Three protein spots were identified by mass spectrometry and validated by Western blotting analysis. To our knowledge, this is the first global quantitation of differential protein expression analysis by 2D-PAGE between LCM cancer cells and normal cells from the same human tumor-tissue sample. Cy3, Cy5, immobilized pH gradient strips, Pharmalyte, and ECL Western blotting detection reagents were purchased from Amersham Biosciences, Inc. Urea and thiourea were bought from Fluka Chemical Corp. (Milwaukee, WI). CHAPS was obtained from Sigma. DTT was purchased from Fisher. Protease-inhibitor mixture tablets were from Roche Molecular Biochemicals. Antibodies used in this study include anti-α-tubulin and all the secondary antibodies (Sigma), anti-annexin 1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-gp96 (StressGen Biotechnology Corp., Victoria, British Columbia, Canada). The SYPRO Ruby staining kit was from Molecular Probes Inc. (Eugene, OR). LCM (19Emmert-Buck M.R. Bonner R.F. Smith P.D. Chuaqui R.F. Zhuang Z. Goldstein S.R. Weiss R.A. Liotta L.A. Laser capture microdissection.Science. 1996; 274: 998-1001Google Scholar) was performed in a Pixcell II laser capture microscope (Arcturus Engineering, Mountain View, CA). Histology of normal and malignant frozen esophageal tissue sections obtained from the same tumor sample was carefully examined by a board-certified pathologist. Frozen sections (8 μm) were stained with hematoxylin, followed by gradual ethanol and xylene dehydration. During the period, proteinase inhibitor mixture tablets were added to the staining solutions. Once air-dried, the section was overlaid with a thermoplastic polymer film mounted on a transparent cap, and targeted cells were captured through focal melting of the membrane by laser pulses. Laser spot size was adjusted to 30 μm, and on average 3–5 cells were collected per laser shot. In general, 250,000 cells were procured for 2D gel electrophoresis, and 20,000–40,000 cells were collected for immunoblotting. As determined by microscopic visualization of the captured cells, the LCM enables isolation of a pure cell population (>95%). 100 μl of isoelectric focusing lysis solution containing 7 m urea, 2 m thiourea, 4% CHAPS, 100 mm DTT, and protease inhibitor mixture was applied directly to the microdissected cells adhered on the LCM cap, which was then placed into an Eppendorf tube and vortexed for all cells were M.A. Sanchez J.C. C. P. Tortola S. Hohenberger W. Kirchner T. Hochstrasser D.F. Kockerling F. sample in colorectal 1997; 18: Scholar, B. T. K. K. A.A. S. G. of human to two-dimensional electrophoresis of Scholar). The isoelectric focusing solution was then to containing cells from the same microdissected and the was each 100 μl the whole cell lysate from cells LCM The cell lysates were in The proteins from normal and cancer cells were then in mm m urea, 4% CHAPS, mm and labeled with of Cy3 and Cy5, to the The labeled samples were and mixed with m urea, 2 m thiourea, CHAPS, m DTT, applied to an 18-cm immobilized pH gradient for isoelectric focusing was carried out on an Amersham Biosciences, Inc. II system as by the immobilized pH gradient pH were used for the for a focusing of The were with a solution containing m urea, mm with 100 mm DTT and directly applied to a gel for electrophoresis at The gel was collected at an excitation of and at an of the gel was collected at an excitation of and an of The resulting gel was then by SYPRO Ruby The SYPRO was at an excitation of and an of the images were collected on a 2D Biosciences, and quantitation of protein expression were carried out in software Biosciences, those spots with changes in between the two were as LCM targeted cells were to the lysis containing m 4% m DTT, The cell lysate was separated on a gel and then to were in in for 1 and with various antibodies for 1 with with mm mm three the were with the secondary antibodies for and developed using the method Biosciences, To the expression of proteins in protein were by Western blotting with the The separated proteins in gels were by SYPRO Ruby The spots of were in-gel and as previously A. Zhao Y. the of a protein in the 2001; 15: Scholar). The sample was in μl of A solution for mass analysis. analysis was performed in an with an system Amersham Biosciences, 2 μl of protein obtained were loaded on the with an To the a high to was applied to the of the through a Inc., The were from the with a gradient of to of in A at a of The were directly from the of the to the mass for mass spectrometry analysis. The was in a the of all in the mass of to and the with the for In this of both the and were The accurately of the and were used to for protein in the protein sequence with the The experimental are as a and are in normal and cancer cells were procured from the same esophageal carcinoma sample. cell lysates were produced and by The and which the resolution of 2D gel analysis A. Postel W. Gunther S. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 1988; 9: 531-546Google Scholar, A. Obermaier C. Boguth G. A. immobilized pH for two-dimensional electrophoresis of and nuclear 1997; 18: Scholar, A. M. T. Y. electrophoresis of proteins and of gel 1996; Scholar). The proteins from both normal cells and cancer cells were labeled with Cy3 and Cy5 dyes, to the The that than of molecules of each protein were labeled. The resulting two pools of proteins were mixed and to isoelectric focusing in a II followed by in the 2D gel images of cancer cells and normal cells were produced in a 2D The of the images can be in on the of protein expression in 2D images were carried out using An analysis of and gels identified 1038 protein spots in cancer cells and 1088 protein spots in normal cells were used as the The of each spot was based on spot and spot and was followed by the with the of all the spots in the gel The of each spot the basis for comparison of protein expression between cancer cells and normal cells As in the the of protein spots a The between cancer and normal was of DIGE to protein expression spot of the Cy3 and Cy5 The the of protein spots in normal cells the those in cancer cells. The protein expression profile between cancer cells and normal cells and to a The was used to the protein expression was between cancer and normal images Of the spots detected in two spots were and in cancer. The of population was with the the of the population was with the protein spots were to be single and protein spots single in cancer cells. Analysis of the protein expression using software of the protein an for the comparison of spot between the two protein spot could be in by amount and The of a protein spot was based on the from the images obtained by the 2D The the of the protein spot in the gel the to the protein of protein spots and could be in by which were from the of the spot by the of the The comparison of the spot is more this way than the conventional way is adjusted to the same As in spot identified as tumor rejection antigen was up-regulated in tumor and in spot identified as annexin I, was down-regulated in tumor Because each was to the for the the spot was by than as in the the protein spots 58 protein spots were up-regulated by >3-fold, and 107 were down-regulated by changes than were protein spots of the detected the quantitation and can be in 30 the of the DIGE To the global changes of protein by dye the same gel was stained with SYPRO The Cy3 and Cy5 originally from the same gel, could be superimposed and directly The patterns between the SYPRO Ruby and the dye images were are subtle differences between the SYPRO Ruby and the which could be by two the molecular mass of the labeled proteins was typically in Cy3 or Cy5 the added to the molecular and the abundance of protein spots in Cy3 or Cy5 images could be either or on the abundance of in the a spot in the SYPRO Ruby in either the Cy5 or Cy3 images A and Because the labeled protein has a molecular mass than the and the minimum number of molecules of each protein were labeled, the protein spot than the labeled protein spot was for mass spectrometry analysis. The protein of or was from the Cy3 or Cy5 and spot in the SYPRO Ruby was The protein spot was and to in-gel and mass analysis in an mass The determined the molecular of from the and which are used to the protein by to protein existing in the protein sequence and sequence The protein identification was by and each was in We are to protein spots that could be by SYPRO Ruby the analysis of of spot of the with a mass of determined the of the A using the mass of the and with software identified sequence to tumor rejection antigen and were identified To the identified proteins were or in a of the protein lysates were to and with the antibodies the protein up-regulated protein tumor rejection antigen and down-regulated protein annexin As was for between cancer cells and normal cells. for the protein a to the molecular mass of the protein was in tumor lysates not in normal cell the of the protein in normal cells. In protein annexin was in normal cells not in tumor cells. Western blotting the from 2D gel analysis and protein identification by mass DIGE is a new approach in differential this technology has not been applied to analysis of cells in human We have successfully applied the DIGE technique to the identification of cancer-specific protein from LCM of human esophageal analysis identified 58 protein spots that were and 107 proteins that were down-regulated than in cancer cells. proteins with than in cancer normal were identified by mass spectrometry and by Western blotting analysis to the differential expression identified by analysis of a human cancer the 2D DIGE technique several the two pools of protein extracts were separated in the same gel, the reproducibility with conventional 2D gel is and the comparison of protein expression patterns is the differences in protein expression between two of proteins can be more accurately and differential protein expression can be based on fluorescence of the labeled Cy3 and Cy5 dyes, providing quantitation of protein Because fluorescence enables quantitation of protein with a dynamic of of A of the of fluorescence detection to two-dimensional gel electrophoresis and 2000; the technique has dynamic than more conventional staining methods such as and DIGE requires to the protein The in DIGE which is than staining methods with detection such as staining and SYPRO Ruby staining DIGE a for high analysis of 2D gels by for gel and quantitation and comparison of gel images and gels are for analysis two pools of samples are separated in the same The technique allows protein quantitation of two pools of protein profiles in than 30 using our The 2D DIGE method high reproducibility. In an not of μg of cell lysates were labeled with Cy3 or Cy5, respectively. 2D DIGE analysis of spots with differences between the spots were in the Cy3 and the two were in the Cy5 were changes gels loaded with the same samples by of proteins by fluorescent not the protein identification by mass Because of the molecules of each protein are labeled, typically not the in mass spectrometry of the and dynamic of mass analysis. Although the technique is a few technical to be the protein patterns obtained with the DIGE system could be from those obtained with conventional The technique on the fluorescence dye for quantitation of of will be for and will be labeled with on The same protein in two pools of protein extracts could be labeled with as as the are those proteins with a high of could be labeled more with proteins containing or In the technique is not to those proteins a high abundance protein spot in a conventional gel system could be a or abundance protein spot in the DIGE system of the changes the of protein spots in a 2D Although the covalently dye will not the of the proteins, the dye molecule has a to the on the it to the molecular mass of the the labeled protein could migrate in in the of the 2D gel than will be more for proteins than for large Because a of the protein is labeled, of the protein molecules cannot be in the Cy3 or Cy5 To the the 2D gel is by SYPRO Ruby and the protein of is identified by comparison of the patterns of the SYPRO and the Cy3 and Cy5 In the of an emerging proteomic 2D DIGE, as demonstrated by the of this tool for profiling and identification of cancer-specific protein using laser capture-microdissected human tissue cells. technique to have of adequate high and a wide dynamic range. technique will be for those applications that quantitation and differential proteomic analysis of normal and cell and large analysis of clinical tissue We and and for for of the DIGE