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• W2106837648 abstract "In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210–2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [35S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFκB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that ∼30% of the 51-member hybrid high abundance CF proteome interacts with the NFκB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine. In previous studies with cystic fibrosis (CF) IB3-1 lung epithelial cells in culture, we identified 194 unique high abundance proteins by conventional two-dimensional gel electrophoresis and mass spectrometry (Pollard, H. B., Ji, X.-D., Jozwik, C. J., and Jacobowitz, D. M. (2005) High abundance protein profiling of cystic fibrosis lung epithelial cells. Proteomics 5, 2210–2226). In the present work we compared the IB3-1 cells with IB3-1/S9 daughter cells repaired by gene transfer with AAV-(wild type)CFTR. We report that gene transfer resulted in significant changes in silver stain intensity of only 20 of the 194 proteins. However, simultaneous measurement of de novo biosynthetic rates with [35S]methionine of all 194 proteins in both cell types resulted in the identification of an additional 31 CF-specific proteins. Of the 51 proteins identified by this hybrid approach, only six proteins changed similarly in both the mass and kinetics categories. This kinetic portion of the high abundance CF proteome, hidden from direct analysis of abundance, included proteins from transcription and signaling pathways such as NFκB, chaperones such as HSC70, cytoskeletal proteins, and others. Connectivity analysis indicated that ∼30% of the 51-member hybrid high abundance CF proteome interacts with the NFκB signaling pathway. In conclusion, measurement of biosynthetic rates on a global scale can be used to identify disease-specific differences within the high abundance cystic fibrosis proteome. Most of these kinetically defined proteins are unaffected in expression level when using conventional silver stain analysis. We anticipate that this novel hybrid approach to discovery of the high abundance CF proteome will find general application to other proteomic problems in biology and medicine. Cystic fibrosis is a common lethal genetic disease that is manifest by intrinsic hyperactivation of proinflammatory signaling pathways in the lung (1Welsh M.J. Ramsey B.W. Accurso F. Cutting G.R. Scriver C.L. Beaudet A.L. Valle D. Sly W.S. Cystic Fibrosis. 8th Ed. McGraw-Hill, New York2001: 5121-5188Google Scholar). The CF airway is massively populated by inflammatory mediators such as IL-8 1The abbreviations used are: IL-8, interleukin 8; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; 2-D, two-dimensional; 2DGE, 2-D gel electrophoresis; AAV, adeno-associated virus; GO, gene ontology; KRT, keratin; HMGCS1, hydroxymethylglutaryl-CoA synthase 1; TCTP, translationally controlled tumor protein; UCHL1, ubiquitin carboxyl-terminal hydrolase 1; IKK, IκB kinase. 1The abbreviations used are: IL-8, interleukin 8; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; 2-D, two-dimensional; 2DGE, 2-D gel electrophoresis; AAV, adeno-associated virus; GO, gene ontology; KRT, keratin; HMGCS1, hydroxymethylglutaryl-CoA synthase 1; TCTP, translationally controlled tumor protein; UCHL1, ubiquitin carboxyl-terminal hydrolase 1; IKK, IκB kinase. that initially come from the lung epithelial cells (2Tirouvanziam R. de Bentzmann S. Hubeau C. Hinnrasky J. Jacquot J. Peault B. Puchelle E. Inflammation and infection in naive human cystic fibrosis airway grafts.Am. J. Respir. Cell Mol. Biol. 2000; 23: 121-127Crossref PubMed Scopus (216) Google Scholar) as well as tumor necrosis factor α, leukotrienes, and others (3Bonfield T.L. Konstan M.W. Burfeind P. Panuska J.R. Hilliard J.B. Berger M. Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin 10, which is downregulated in cystic fibrosis.Am. J. Respir. Cell Mol. Biol. 1995; 13: 257-261Crossref PubMed Scopus (326) Google Scholar, 4Bonfield T.L. Panuska J.R. Konstan M.W. Hilliard K.A. Hilliard J.B. Ghnaim H. Berger M. Inflammatory cytokines in cystic fibrosis lungs.Am. J. Respir. Crit. Care Med. 1995; 152: 2111-2118Crossref PubMed Scopus (597) Google Scholar). Although it has been long known that CF is due to mutations in the CFTR gene (5Riordan J.R. Rommens J.M. Karem B.-S. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Drumm M.L. Iannuzzi M.C. Collins F.S. Tsui L.-C. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA.Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5858) Google Scholar, 6Rommens J.M. Iannuzzi M.C. Karem B.-S. Drumm M.L. Dean M. Rozmahel R. Cole J.L. Kennedy D. Hidaka N. Zciga M. Buchwald M. Riordan J.R. Tsui L.-C. Collins F.S. Identification of the cystic fibrosis gene: chromosome walking and jumping.Science. 1989; 245: 1059-1065Crossref PubMed Scopus (2494) Google Scholar, 7Karem B.-S. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L.-C. Identification of the cystic fibrosis gene: genetic analysis.Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3161) Google Scholar), the mechanism linking the CFTR mutation to inflammatory disease is still not known. Recent pharmacogenomic and pharmacoproteomic studies have implicated defects in both the TNFα/NFκB signaling pathway (8Eidelman O. Srivastava M. Zhang J. Murthy J. Heldman E. Jacobson K.A. Metcalfe E. Weinstein D. Pollard H.B. Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-αR/NFκB pathway.Mol. Med. 2001; 7: 523-534Crossref PubMed Google Scholar, 9Srivastava M. Eidelman O. Zhang J. Paweletz C. Caohuy H. Yang Q.-F. Jacobson K.A. Heldman E. Catherine Jozwik C. Pollard B.S. Pollard H.B. Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells.Proc. Nat. Acad. Sci. U. S. A. 2004; 101: 7693-7698Crossref PubMed Scopus (76) Google Scholar, 10Yang Q.-F. Huang W. Jozwik C. Lin Y. Glasman M. Caohuy H. Srivastava M. Pollard H.B. Cardiac glycosides inhibit TNFα/NFκB signaling by blocking recruitment of TRADD to the TNF receptor.Proc. Nat. Acad. Sci. U. S. A. 2005; 102: 9631-9636Crossref PubMed Scopus (79) Google Scholar, 11Tchilibon S. Zhang J. Yang Q.F. Eidelman O. Kim H. Caohuy H. Kenneth A. Jacobson K.A. Pollard B.S. Pollard H.B. Amphiphilic pyridinium salts suppress production of the proinflammatory cytokine interleukin-8 in cystic fibrosis lung epithelial cells.Biochem. Pharmacol. 2005; 70: 381-393Crossref PubMed Scopus (21) Google Scholar) and the HSP70 system (12Wright J.M. Zeitlin P.L. Cebotaru L. Guggino S.E. Guggino W.B. Gene expression profile analysis of 4-phenylbutyrate treatment of IB3-1 bronchial epithelial cell line demonstrates a major influence on heat-shock proteins.Physiol. Genomics. 2004; 16: 204-211Crossref PubMed Scopus (70) Google Scholar). However, parallels between changes in mRNA and cognate proteins are infrequent in eukaryotic cells principally because of many overlapping layers of post-transcriptional and post-translational regulation.We have therefore embarked on a global approach to the CF proteome in cultured CF lung epithelial cells and in primary cultures of bronchial lung epithelium obtained by brush biopsy of CF patients. Using an approach of 2-D gel electrophoresis (2DGE) and mass spectrometry, we recently identified 194 unique high abundance proteins in the CF lung epithelial IB3-1 cell system (13Pollard H.B. Ji X.-D. Jozwik C.J. Jacobowitz D.M. High abundance protein profiling of cystic fibrosis lung epithelial cells.Proteomics. 2005; 5: 2210-2226Crossref PubMed Scopus (31) Google Scholar). Preliminary comparisons between the IB3-1 cells and IB3-1/S9 cells, a daughter cell line repaired by gene transfer with wild type CFTR, revealed that relatively few changes in silver-stained features could be detected for any of the 194 high abundance proteins. We therefore used [35S]methionine to measure the de novo biosynthetic rates of each of the 194 proteins we had previously identified on the 2-D gel format. The advantage of radiolabeling is that an entire proteome can be visualized at once from a phosphorimage or autoradiograph, and experimental differences can be immediately annotated. We report here that there is significant proteomic information that can be obtained by simultaneously measuring the individual mass levels and de novo biosynthetic rates for all identified proteins. We conclude that de novo biosynthetic labeling can be used in combination with conventional mass labeling to identify disease-specific differences within the high abundance cystic fibrosis proteome. These differences can be used to generate a hybrid high abundance proteomic signature for cystic fibrosis.EXPERIMENTAL PROCEDURESCultured Cells and Reagents—The CF lung epithelial cells IB3-1 and AAV-(wild type)CFTR-repaired IB3-1/S9 have been described previously (Refs. 8Eidelman O. Srivastava M. Zhang J. Murthy J. Heldman E. Jacobson K.A. Metcalfe E. Weinstein D. Pollard H.B. Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-αR/NFκB pathway.Mol. Med. 2001; 7: 523-534Crossref PubMed Google Scholar, 9Srivastava M. Eidelman O. Zhang J. Paweletz C. Caohuy H. Yang Q.-F. Jacobson K.A. Heldman E. Catherine Jozwik C. Pollard B.S. Pollard H.B. Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells.Proc. Nat. Acad. Sci. U. S. A. 2004; 101: 7693-7698Crossref PubMed Scopus (76) Google Scholar, and 14Eidelman O. Guay-Broder C. van Galen P.J.M. Jacobson K.A. Turner R.J. Cabantchik Z.I. Pollard H.B. A1 adenosine-receptor antagonists activate chloride efflux from cystic fibrosis cells.Proc. Nat. Acad. Sci. U. S. A. 1992; 89: 5562-5566Crossref PubMed Scopus (91) Google Scholar; see supplemental materials, SM.1). Both IB3-1 and IB3-1/S9 cells were grown in serum-free LHC-8 medium (Biofluids, Bethesda, MD; see supplemental materials, SM.2). The high IL-8 secretion phenotype was routinely monitored by ELISA (R&D Systems, Boston, MA). To avoid potential problems with clonal drift, new batches of frozen cell stocks were thawed at least every 3 months (after ∼12 passages) during the course of the experiment.The proteomic radiolabeling protocol for cells was as follows. Briefly CF cells (“IB3-1” or explants from bronchial biopsies) and repaired cells (“IB3-S9”) were grown in T75 flasks to 80% confluence. To begin the labeling process, cultures were first incubated for 30 min in methionine-free, gentamycin-free LHC-8 medium. Cultures were then incubated in the same medium supplemented with 200 μCi/ml [35S]methionine (PerkinElmer Life Sciences) for an additional 30 min. Preliminary studies over longer time courses had indicated that the 30-min time point was in the linear portion of the biosynthetic process for high abundance proteins. The cells were then washed, and total proteins were prepared for 2-D gel electrophoresis. The entire analysis was performed on a total of 30 independently performed experiments, in triplicate, featuring both IB3-1 and IB3-1/S9 cells in parallel. Additional radiolabeling information is given in the supplemental materials (SM.3).Human Subjects—CF and non-CF individuals who were undergoing a bronchoscopy for a clinical indication were eligible. Patients (or parents on behalf of a person <18 years of age) signed informed consent for an Institutional Review Board- and General Clinical Research Center-approved protocol to obtain bronchial specimens. An additional assent form was signed by adolescent participants. Bronchoscopy was performed under general anesthesia. Bronchial brushings were obtained from second or third generation airways under direct visualization and immediately immersed in ice-cold gentamycin-supplemented LHC-8 medium. Bronchoalveolar lavage was performed according to the Cystic Fibrosis Therapeutics Development Network standard operating procedure. There were no significant or serious adverse events associated with specimen collection.2DGE and Mass Spectrometry—Epithelial cells were grown in the appropriate media to ∼6 × 106 cells/100-mm tissue culture dish (∼80% confluence). At the conclusion of the incubation period, 300 μl of lysis buffer (8 m urea, 4% CHAPS, 40 mm Tris-Cl, pH 8.5) were added to the culture dish, and the lysed cells were collected by scraping the dish with a cell lifter (Corning Inc., Corning, NY). The lysates were sonicated briefly and centrifuged at 20,000 × g at 4 °C for 10 min, and the supernatant was stored at −70 °C. Protein concentrations were determined using the BCA assay kit (Pierce). Two-dimensional gel electrophoresis of 100–200 μg of protein was performed exactly as described previously (Ref. 13Pollard H.B. Ji X.-D. Jozwik C.J. Jacobowitz D.M. High abundance protein profiling of cystic fibrosis lung epithelial cells.Proteomics. 2005; 5: 2210-2226Crossref PubMed Scopus (31) Google Scholar; see supplemental materials, SM.4). Images of silver-stained 2-D gels were obtained using the Fujifilm Intelligent Dark Box II LAS-1000 imaging system (Fujifilm, Stamford, CT). Proteins that had incorporated [35S]methionine were imaged using a Typhoon phosphorimaging system (Molecular Probes, Mountain View, CA). A complete description is given in the supplemental materials, SM.4 and SM.5.Bioinformatics and Statistics—Thirty independent experiments comparing cultures of IB3-1 and AAV-(wild type)CFTR IB3-1/S9 cells were used for the analysis. Each experiment compared 100, 200, and 300 μg of total protein in triplicate. Intensities of silver-stained features were routinely corrected by using the normalization function of the ImageMaster software. This normalization is operationally equivalent to linear fitting of the log intensities to the inverse normal function of the rank order of the intensities (see supplemental materials, SM.6). This approach to normalization gives a linear fit to the data except for the very high range (where saturation effects prevail) and the very low range (where low signal-to-noise effects predominate). This strategy therefore corrects the lowest intensities and highest intensities, respectively, by extrapolating the linear fit. Equivalent protein features on different gels were identified, and their fractional volumes were calculated using the ImageMaster software. A false discovery rate of <10%, calculated by Significance Analysis of Microarrays (www-stat.stanford.edu/∼tibs/SAM), was taken to be significant (see supplemental materials, SM.7). Identification of proteins was accomplished by mass spectrometry of protein spots punched from the gels, coincident with acquisition of data for our recently published atlas (13Pollard H.B. Ji X.-D. Jozwik C.J. Jacobowitz D.M. High abundance protein profiling of cystic fibrosis lung epithelial cells.Proteomics. 2005; 5: 2210-2226Crossref PubMed Scopus (31) Google Scholar). We also used pattern matching ImageMaster™ 2G Platinum software to correlate spots on one gel with cognate spots on others (see supplemental materials, SM.8). Protein identification of six samples, in addition to those identified in Ref. 13Pollard H.B. Ji X.-D. Jozwik C.J. Jacobowitz D.M. High abundance protein profiling of cystic fibrosis lung epithelial cells.Proteomics. 2005; 5: 2210-2226Crossref PubMed Scopus (31) Google Scholar, were validated on the Thermo-Finnegan LCQ Deca XP electrospray ion trap mass spectrometer (Thermo-Electron Corp., West Palm Beach, FL) and on the Q-Star XL electrospray quadrupole time-of-flight mass spectrometer (Applied Biosystems, Natick, MA) (15Elias J.E. Haas W. Faherty B.K. Gygi S.P. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations.Nat. Methods. 2005; 2: 667-675Crossref PubMed Scopus (600) Google Scholar). The hierarchical clustering algorithm was utilized as described previously for application to genomic analysis (Refs. 8Eidelman O. Srivastava M. Zhang J. Murthy J. Heldman E. Jacobson K.A. Metcalfe E. Weinstein D. Pollard H.B. Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-αR/NFκB pathway.Mol. Med. 2001; 7: 523-534Crossref PubMed Google Scholar and 9Srivastava M. Eidelman O. Zhang J. Paweletz C. Caohuy H. Yang Q.-F. Jacobson K.A. Heldman E. Catherine Jozwik C. Pollard B.S. Pollard H.B. Digitoxin mimics gene therapy with CFTR and suppresses hypersecretion of IL-8 from cystic fibrosis lung epithelial cells.Proc. Nat. Acad. Sci. U. S. A. 2004; 101: 7693-7698Crossref PubMed Scopus (76) Google Scholar; see supplemental materials, SM.9). Expression levels in cells and tissues were analyzed by non-parametric statistics (see supplemental materials, SM.6). Gene ontologies were analyzed by GOMiner software (see supplemental materials, SM.10). For analysis of functional or physical connectivity among identified proteins, we used the PathwayAssist databases and software (Ariadne Genomics, Inc.) (Ref. 16Nikitin A. Egorov S. Daraselia N. Mazo I. Pathway studio—the analysis and navigation of molecular networks.Bioinformatics. 2003; 19: 2155-2157Crossref PubMed Scopus (509) Google Scholar; see supplemental materials, SM.11).RESULTSParallel Silver Staining and Radiolabeling of CF IB3-1 and Repaired IB3-1/S9 Cells—As shown in Table I and Fig. 1, high abundance proteins from CF lung epithelial IB3-1 cells (17Zeitlin P.L. Lu L. Hwang T.-C. Rhim J. Cutting G.R. Keiffer K.A. Craig R. Guggino W.B. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno12-SV40 infection.Am. J. Respir. Cell Mol. Biol. 1991; 4: 313-319Crossref PubMed Scopus (273) Google Scholar) and their AAV-(wild type)CFTR-repaired daughter cell line IB3-1/S9 (18Egan M. Flotte T. Afione S. Solow R. Zeitlin P.L. Carter B.J. Guggino W.B. Defective regulation of outwardly rectifying Cl− channels by protein kinase A corrected by insertion of CFTR.Nature. 1992; 358: 581-584Crossref PubMed Scopus (377) Google Scholar) can be compared by simultaneous imaging with either silver stain or incorporation of [35S]methionine. Fig. 1, a and b, shows a comparison of silver-stained images of CFTR-repaired and parental CF cells, respectively. There were substantial similarities, as might be anticipated, as well as significant but fewer optical density differences. Below each silver-stained gel, Fig. 1, c and d, shows the same gels, respectively, but imaged according to incorporation with [35S]methionine. There appear to be many more radiolabeled features than silver-stained features. The numbers and intensities of radiolabeled spots increased with exposure time. Most silver-stained spots had some associated radiolabel, whereas many radiolabeled features were devoid of silver stain. Apparently many of the low abundance proteins, otherwise hidden by the relative insensitivity of the silver stain paradigm, can be detected by highly sensitive de novo biosynthesis.Table IProteins significantly differentially expressed in IB3-1 relative to IB3-1/89View Large Image Figure ViewerDownload Hi-res image Download (PPT)[CODE: ▴=up; ▾=down; ▪=no change] Open table in a new tab Fig. 1Parallel analysis of CF lung epithelial IB3-1 and AAV-(wild type)CFTR-repaired IB3-1/S9 cells by silver stain and biosynthetic labeling with [35S]methionine.a, proteins from (wild type)CFTR-repaired IB3-1/S9 cells are separated on a pH 4–7 2DGE format. Proteins are imaged by silver staining. b, proteins from CF IB3-1 cells are separated on a pH 4–7 2DGE format. Proteins are imaged by silver staining. c, proteins from (wild type)CFTR-repaired IB3-1/S9 cells incubated for 30 min with [35S]methionine and separated on a pH 4–7 2DGE format. Proteins are imaged by autoradiography. d, proteins from CF IB3-1 cells incubated for 30 min with [35S]methionine and separated on a pH 4–7 2DGE format. Proteins are imaged by autoradiography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 2 shows a detailed comparison of a portion of the gels from Fig. 1 as imaged by either silver or radiolabel. Silver stain at low resolution is shown in the upper left, and radiolabel of the same region at low resolution is shown in the upper right. The acutely truncated silver-stained peaks emphasize the narrow dynamic range of silver staining. Operationally these silver-stained features saturate out quickly as a function of mass. The radiolabeled features show graded peaks, consistent with the 5-log dynamic range characteristic of radioactivity. Radiolabeled peaks therefore saturate out slowly as a function of radiolabel. Also there are additional features that are otherwise not apparent in the companion silver-stained image. The lower images in Fig. 2 (a and b) are equivalent expanded views of the same regions that are either silver-stained or radiolabeled. The many additional radiolabeled features can be appreciated when compared with the companion expanded silver-stained image. Importantly most of the radiolabeled features that occurred in the absence of a companion silver-stained image lacked sufficient mass of protein to permit identification by conventional mass spectrometry.Fig. 2Detail from three-dimensional rendering of 2DGE of IB3-1 cell proteome.Silver represents local features detected by silver stain. [35S]Met represents the same local region as detected by radiolabel. a, low magnification image of a region from a silver-stained 2-D gel. b, low magnification image of the same region as in a but imaged by 35S radiolabel. c, high magnification from the circled domain in a, silver stain. d, high magnification from the circled domain in b, radiolabel. The radiolabeled scan reveals ∼5-fold more features than the silver-stained scan. The peaks of the radiolabeled features are graded compared with the chopped off forms of the silver-stained features.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative Comparison of Silver-stained and Radiolabeled Features between CF IB3-1 and Repaired IB3-1/S9 Cells—Thirty different experiments with IB3-1 and repaired IB3-1/S9 cells were performed over a 2-year period in which cells were pulsed with [35S]methionine and the proteins were separated on 2-D gels. The features visible by radiolabel were identified by superposition with known proteins as identified in the IB3-1/S9 proteomic atlas (13Pollard H.B. Ji X.-D. Jozwik C.J. Jacobowitz D.M. High abundance protein profiling of cystic fibrosis lung epithelial cells.Proteomics. 2005; 5: 2210-2226Crossref PubMed Scopus (31) Google Scholar). Fig. 3 shows a comparison between individual protein masses (silver stain volumes) of 194 specific proteins in repaired IB3-1/S9 cells with the cognate proteins in the CF IB3-1 cells. Error bars for different proteins are based on analysis of all data. The filled red circles identify those proteins that differed between IB3-1 and IB3-1/S9 cells at the p < 0.05 level of significance. An equivalent comparison of radiolabeled features can be seen in Fig. 4. The distributions show that cognate radiolabeled proteins were distributed in similar fashion in both CF IB3-1 and repaired IB3-1/S9 cells. Here again the filled red circles indicate proteins whose differences were significant at the p < 0.05 level. Importantly the significantly different silver-stained proteins were infrequently identical to significantly different radiolabeled proteins (see comparisons in Table I).Fig. 3Comparison of intensities of silver-stained features from CF lung epithelial IB3-1 with equivalent silver-stained spots on AAV-(wild type)CFTR-repaired IB3-1/S9 cells.Filled red circles denote proteins showing significant differences between cells at the p < 0.05 level.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Comparison of intensities of radiolabeled features from CF lung epithelial IB3-1 with equivalent radiolabeled features on AAV-(wild type)CFTR-repaired IB3-1/S9 cells.Filled red circles denote proteins showing significant differences between cells at the p < 0.05 level.View Large Image Figure ViewerDownload Hi-res image Download (PPT)A comparison of silver-stained feature volumes and radiolabeled feature volumes is shown in Fig. 5. Although approximately linear, the R2 value of this graph is visibly and analytically much greater than the individual comparisons of IB3-1 and IB3-1/S9 cells in Figs. 3 and 4. Clearly even for the IB3-1 cell alone, the data for silver-stained and radiolabeled proteins demonstrate substantial disparity.Fig. 5Comparison of intensities of silver-stained features from CF lung epithelial IB3-1 with equivalent radiolabeled features.Filled red circles denote proteins showing significant differences between cells at the p < 0. 05 level.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of Silver-stained and Radiolabeled CF Proteomes Using a Hierarchical Clustering Algorithm—To investigate the informational basis of the substantial disparity between silver-stained and radiolabeled features in the IB3-1 cell system, we analyzed all 30 experiments using a hierarchical clustering algorithm. As shown in Fig. 6, this analytic approach clearly permitted the discrimination between IB3-1 and repaired IB3-1/S9 cells. Furthermore the analysis showed a clear discrimination between silver-stained and radiolabeled features. As with all displays of this sort, red marks are high values, and green marks are low values. The horizontal axis on the top of the image shows a primary dyad that separates silver from radiolabel and a secondary dyad that separates IB3-1 from IB3-1/S9 cells. On the vertical axis of identified proteins, the primary dyad distinguishes between an upper cluster of proteins with lower silver staining and higher [35S]methionine incorporation from a lower cluster of proteins with higher silver staining and lower 35S incorporation. The upper cluster of low total mass proteins appears to be more biosynthetically active than the lower cluster of high mass proteins. Individual subdyads connecting various proteins identify those high abundance proteins whose expressions are coupled as a function of the CFTR mutation.Fig. 6Hierarchical clustering algorithm for comparison of IB3-1 and IB3-1/S9 cells, comparing silver staining and radiolabeling of features on the 2DGE. Triplicate data from 30 independent experiments are clustered in terms of silver stain intensities and de novo rates of [35S]methionine incorporation. Classes of proteins can be identified that discriminate between lower abundance proteins that are more rapidly labeled and higher abundance proteins that are more slowly labeled proteins. (Data for this calculation are given in the supplemental materials, SM.12.)View Large Image Figure ViewerDownload Hi-res image Download (PPT)Further ImageMaster analysis of the proteins also allowed us to identify a subcategory of 51 CF-specific proteins whose expression of either total level or biosynthetic rate differed significantly (false discovery rate <10%) when comparing IB3-1 cells with (wild type)CFTR-repaired IB3-1/S9 cells. As shown in Table I, we" @default.
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• W2106837648 title "De Novo Biosynthetic Profiling of High Abundance Proteins in Cystic Fibrosis Lung Epithelial Cells" @default.
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