The Optimization of Protocols for Proteome Difference Gel Electrophoresis (DiGE) Analysis of Preneoplastic Skin Adele Hannigan, Richard Burchmore, and Joanna B. Wilson* Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, United Kingdom Received December 20, 2006
Difference gel electrophoresis (DiGE) allows the reliable comparison of proteome differences between two or three samples within a single gel, by way of a CyDye fluorescent labeling system. This facilitates identification of protein differences avoiding the difficulties associated with gel-to-gel variation. A drawback of this approach is the necessity for high-purity protein samples, since contaminants can interfere with the labeling process, affecting subsequent analysis. Thus far, DiGE has been applied to the study of various sample types derived from relatively simple starting materials such as serum, cell lines, or primary cells. Herein, we describe optimization of protein extraction and purification from a complex tissue (the murine ear) of which a major component is skin, which is compatible with the CyDye labeling system and DiGE. Protein samples obtained by this method from preneoplastic, transgenic tissue have been effectively compared to normal tissue samples to reveal bona fide differences, verifiable by Western blotting. In total, 41 protein differences (21 up- and 20 down-regulated in the pathological samples) were identified by mass spectrometry (MS). This method can therefore form a guide for those wishing to perform DiGE on complex tissues, and is especially useful for samples with relatively insoluble components such as skin. Keywords: DiGE • skin • proteomics • epidermis • transgenic • hyperplasia
Introduction DiGE is a means by which different protein samples can be compared on the same gel and was first described in 19971 and reviewed in ref 2. This is accomplished by labeling the protein samples with different fluorophores, commonly the three CyDyes, Cy5, Cy3, and Cy2, which are size- and chargematched and minimally label the lysine residues of the proteins. The analysis of differential expression levels relies on the difference in intensity between Cy3 and Cy5 fluorescence. Cy2 can be used to label a normalizing control sample which can be included in experimental replicates or to increase the number of comparisons. The protein samples to be labeled must be free from any contaminants that can interfere with the labeling process. Crude lysates produced by mechanical disruption of biological material typically contain a diversity of non-protein macromolecules and salts that can compete for CyDye labeling. The most efficient method to remove such interfering components is to precipitate protein samples prior to the labeling reaction. To examine the proteome changes by DiGE during the early stages of carcinogenesis, a transgenic mouse model was used in which expression of the Epstein-Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), has been directed to epithelia.3,4 A nasopharyngeal carcinoma derived EBV strain * Corresponding author. Tel, 0141 330 5108; e-mail, Joanna.Wilson@ bio.gla.ac.uk.
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variant, termed LMP1CAO, was used. The most striking phenotype presents in the ears of the L2LMP1CAO mice, where transgene expression causes a progressive pathology, from initial mild hyperplasia with increased vascularization to severe hyperplasia with necrosis and tissue degeneration leading to keratoacanthosis and occasional carcinoma.4 The preneoplastic phenotype has been categorized into five recognizable and predictable stages, providing an excellent system in which to study changes in protein expression through the early stages of carcinogenesis. DiGE has been applied to the study of several cell types and tissues including mouse brain tissue, human serum, human liver cells, and rat liver tissue.5-9 However, to date, there are no reports on the application of DiGE to study a stratified epithelial tissue and in particular skin, with the relatively insoluble cornified layer and, hence, how to prepare extracts compatible with the CyDye labeling process. Leonard et al.9 extracted protein from whole rat liver but lyophilized and centrifuged the samples instead of precipitating the proteins prior to labeling, which would likely be problematic if applied to stratified epithelial tissues. DiGE has been applied to the study of a number of mucosal epithelial tissues and cells, including a comparative analysis of oesophageal cancer cells with normal oesophageal cells.10 The cells were isolated by laser capture microdissection, and therefore, the samples were effectively cell-only and were not complicated by the presence of connective tissue, vasculature, and cartilage. Conventional 10.1021/pr0606878 CCC: $37.00
2007 American Chemical Society
research articles
Optimization for DiGE
two-dimensional gel electrophoresis (2D-GE) has been extensively applied to the study of skin proteins, in particular, keratin protein expression. Furthermore, the Danish center for translational breast cancer research database holds proteome maps of keratinocytes and carcinomas,11 and recently, Huang et al. have described their efforts to generate a proteome map of murine skin using the BALB/c mouse strain.12,13 Moreover, a study has been conducted comparing punch skin biopsies (predominantly epidermis) of elderly and young donors.14 Thus, there are a number of reports describing protein extraction from whole skin tissue; however, these were not purified by precipitation and were therefore not compatible with a CyDye labeling protocol. Herein, we describe the comparison of a variety of protein extraction buffers and precipitation techniques, the optimal of which was used to conduct a comparative proteomic analysis of a whole tissue, murine ear, of which skin is a major component. The design of the DiGE experiments, including choice of pH range and the validity and confirmation of the results by Western blot, is discussed.
Experimental Procedures Transgenic Mice. L2LMP1 (or L2LMP1CAO) transgenic mice of line 117 and line 105B (the latter expressing the transgene at a lower level compared to the former) were used in these studies and compared to non-transgenic sibling controls (NSC), thereby ensuring consistency in mouse age, strain, and environmental conditions.4 The mice were maintained in the FVB strain and housed under conventional conditions. All procedures have been conducted under UK Home Office license, and the research has complied with Home Office and institutional guidelines and policies. Tissue samples were snap-frozen in liquid nitrogen and stored at -70 °C. Sample Preparation. Extraction of protein from ear tissue was performed using one of five buffers (Table 1). The tissue was cut into fine pieces and homogenized using a polytron directly in extraction buffer (approximately 100 mg tissue/mL) containing 20 µL/mL protease inhibitors (complete mini, Roche). The homogenized samples were freeze/thawed using liquid N2 twice and sonicated for 10 s, incubated on ice for 15 min and insoluble matter removed by centrifugation. Aliquots (150 µL) were precipitated with one of the following precipitation methods: (i) 2D clean up kit (GE Healthcare, as per manufacturer’s instructions). (ii) TCA precipitation. Trichloroacetic acid (TCA; 100% (w/v) in dH2O) was added to the protein extract to a final concentration of 15% (w/v) TCA. The sample was incubated on ice for 45 min and centrifuged at 14 000g for 5 min at 4 °C, and the supernatant was removed. The resulting pellet was washed three times with ice-cold acetone and centrifuged at 14 000g, 5 min at 4 °C. (iii) TCA/acetone precipitation. Three volumes of 13.3% (w/v) TCA in acetone (0.07% (w/v) 2-mercaptoethanol) was added to the extract. The precipitate was formed at -20 °C for 45 min and centrifuged at 14 000g for 5 min at 4 °C, and the supernatant was removed. The resulting pellet was washed three times with ice-cold acetone and 0.07% (w/v) 2-mercaptoethanol and centrifuged at 14 000g for 5 min at 4 °C. (iv) Acetone precipitation. Four volumes of ice-cold acetone was added to the extract, and the precipitate was formed at -20 °C for 1.5 h. The sample was centrifuged at 14 000g for 15 min at 4 °C, and the supernatant was removed. The resulting pellet was washed twice with 80% (v/v) ice-cold acetone and centrifuged at 14 000g for 5 min at room temperature. (v) Ammonium acetate precipitation. Five volumes of 0.1 M ammonium acetate in methanol was added
Table 1. Protein Extraction and Purification for DiGEa extraction buffer
precipitation protocol
1 8 M urea, 30-40 mM Tris, pH 8-8.5, 4% (w/v) CHAPS 2 175 mM Tris, pH 8, 5% (w/v) SDS, 15% (v/v) glycerol 3 50 mM Tris, pH 8, 150 mM NaCl, 5mM EDTA, 1% (v/v) NP40 4 8 M urea, 10 mM Tris, pH 8, 5 mM Mg(Ac)2, 2% (w/v) CHAPS 5 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 30 mM Tris, pH 8.5 extraction Yield of precipitation % recovery buffer proteinb protocol of proteinc
1
+++
i
94%
1
+++
ii
19%
1 1
+++ +++
iii iv
16% 66%
1
+++
v
69%
2
++++
i
53%
2
++++
iv
nt
3 4 5
+++ +++ ++
iv v i
nt 54% 97%
i
2D clean up kit
ii
TCA
iii TCA/acetone iv acetone v
ammonium acetate
quality of 2D image
resolution reasonable good number of spots horizontal streaking, poor resolution nt horizontal streaking, poor resolution resolution and spot number reasonable very good resolution of spots and high number horizontal streaking, low spot number nt low number of spots resolution and spot number reasonable
a The top panel details the five extraction buffers used and the five precipitation methods. The bottom panel shows how these were used in conjunction. b The relative yield of protein directly after extraction with the different buffers (low, + to high, ++++). c The % recovery of protein, comparing quantity before and after precipitation. nt ) not tested.
to the extract, and the proteins were precipitated at -20 °C overnight. The sample was then centrifuged at 14 000g for 10 min at 4 °C, and the supernatant was removed. The resulting pellet was washed twice with 80% (0.1 M ammonium acetate in methanol), three times with 80% (v/v) acetone and centrifuged at 14 000g for 2 min at 4 °C. In all cases, the pellet was briefly air-dried prior to resuspension. The precipitates were resuspended in 7 M urea, 2 M thiourea, 2% (w/v) CHAPS, and 30 mM Tris. Protein concentration was determined using the 2D Quant kit (GE Healthcare) as per the manufacturer’s instructions. Standard 2D SDS-PAGE Minigels. A total of 50 µg of protein extract was made up to 125 µL with rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, and trace bromophenol blue) containing 0.5% (v/v) of the appropriate IPG buffer (GE Healthcare) and 10 mg/mL DTT. This was distributed evenly over the base of a 7 cm Immobiline DryStrip holder. The gel side of a 7 cm IPG strip was placed on top of the solution, avoiding bubbles, and the strip backing was covered with PlusOne DryStrip cover fluid, ∼175 µL. The holders were placed on an Ettan IPGphor IEF unit and focused overnight; rehydration, 30 V for 14 h; IEF, 500 V for 1 h, 1000 V for 1 h, 5000 V for 4 h, for a total of approximately 22 000 Vh. The strips were equilibrated for 15 min in 10 mL of SDS-equilibration buffer (75 mM Tris, pH8.8, 6 M urea, 2% (w/v) SDS, and trace of bromophenol blue) containing 10 mg/mL DTT, followed by 15 min in 10 mL of SDS-equilibration buffer containing 25 mg/mL R-iodoacetamide. The strip was then applied to the top of a 12% acrylamide minigel and electrophoresed at 100 V in Journal of Proteome Research • Vol. 6, No. 9, 2007 3423
research articles running buffer (192 mM glycine, 25 mM Tris, and 0.2% (w/v) SDS) until the dye front reached the gel’s end.5 The gels were fixed in 10% (v/v) acetic acid and 40% (v/v) ethanol for 3 h, washed twice for 10 min in ddH2O, and then stained in colloidal coomassie stock (10% (w/v) ammonium sulfate, 1.2% (v/v) o-phosphoric acid, 0.1% (w/v) coomassie blue G250)/methylated spirits (4:1) for 1-7 days and de-stained in ddH2O. DiGE. Protein was extracted using buffer 2 (Table 1), precipitated with the 2D clean up kit (protocol i), and quantified using the 2D Quant kit. The pH of each sample was adjusted to ∼pH 8.5 with 50 mM NaOH if required. Samples were aliquoted at 50 µg, and the pooled internal standard was made with 31.25 µg of each of the eight test samples combined. The proteins were labeled with 400 pmol (in 1 µL of anhydrous DMF) of CyDye per 50 µg of protein as per the manufacturer’s instructions (GE Healthcare). After labeling, the appropriate samples were combined for each gel. To each combined sample was added 1 mg of DTT and 2.25 µL of IPG buffer and then made up to 450 µL with rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, and trace bromophenol blue). The samples were mixed and pipetted onto the base of a 24 cm strip holder, an immobiline DryStrip (pH 4-7 or pH 6-9) was placed on top, and the backing of the strip was covered with ∼900 µL of DryStrip cover fluid (GE Healthcare). The strips were focused as follows: rehydration step, 30 V, 10 h (minimum); step-n-hold, IEF, 300 V, 2 h; step-n-hold, 600V, 2 h; gradient, 1000 V, 2 h; gradient, 8000 V, 3 h; gradient, 8000 V, 3 h; stepn-hold, with current limited to a maximum of 50 µA/strip throughout the protocol. The IPGphor IEF unit was covered to exclude light and the strips were focused for approximately 70 000Vh. After focusing, the strips were equilibrated in SDSequilibration buffer plus 10 mg/mL DTT (15 min), then 25 mg/mL R-iodoacetamide (15 min) as detailed above. The immobiline DryStrips were applied to the tops of 12.5% polyacrylamide gels and electrophoresed at ∼100 V overnight in running buffer.5 After electrophoresis, one of the gels was prescanned using the Typhoon 9400 variable mode imager at each of the appropriate CyDye excitation wavelengths (Cy3 (532 nm), Cy5 (633 nm), Cy2 (488 nm)), in order to determine the appropriate laser intensity for each that would generate robust images without any spot saturation. Thereafter, each of the analytical gels was scanned at this optimum laser intensity. A preparative gel was also prepared, which was loaded with 400 µg of unlabeled protein (200 µg each of transgenic and NSC sample), together with 50 µg of the Cy2-labeled pooled standard. This gel was scanned using a 488 nm excitation (Cy2) only, then fixed and stained with SYPRO orange (Sigma, as per manufacturer’s instructions) and re-scanned using the 532 nm laser. Scanned images were analyzed using DeCyder software modules. Spot-Picking and Mass Spectrometry. This was conducted essentially as described in ref 5 picking spots from preparative gels. Western Blotting. Ears were homogenized in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% (v/v) Triton X-100, 1% (w/v) deoxycholic acid, and 0.1% (w/v) SDS) containing 20 µL/mL protease inhibitors (Roche) and 10 µL/mL phosphatase inhibitor cocktail (Sigma), incubated on ice for 15 min and insoluble matter removed by centrifugation. Aliquots (5 and 10 µL) were used to determine the protein concentration of the sample using the 2D Quant assay. Proteins (40 µg per track) were separated by SDS-PAGE (7.5% or 12.5%) and blotting and washing was performed as previously described.15 The blots 3424
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were incubated in 5% non-fat milk PBS and 0.1% (v/v) Tween 20 with either 1:1000 anti-keratin 6 (PRB-169P, Convance), 1:200 anti-superoxide dismutase 1 (sc-11407, Santa Cruz), 1:1000 anti-keratin 1 (PRB-165P, Convance), and 1:2000 antiGAPDH 6C5 (H86504M, Biodesign), followed by 1:4000 of the appropriate IgG-HRP-conjugated secondary antibody (antirabbit sc-2030, anti-mouse sc-2031, Santa-Cruz) and visualized by enhanced chemiluminescence (liteAblot kit, Euroclone).
Results Preparation of Proteins for DiGE. Proteins were extracted from whole ears of L2LMP1 transgenic mice phenotypically categorized at stage 2 or stage 5 along with negative sibling controls (NSC) using one of five alternative buffers (Table 1) and precipitated using one of five protocols (the latter assessed at least three times each apart from precipitation iii) (Table 1). The protocols were assessed for the yield of protein, the percent recovery after precipitation, and the quality of the 2D gel image (Table 1). Only protein samples from conditions yielding a suitably high quantity of protein after precipitation (>50%) were selected for 2D gel analysis. The proteins were focused on 7 cm pH 4-7, immobilized pH gradient (IPG) strips, electrophoresed on small 12% acrylamide gels, and visualized using colloidal coomassie blue staining. Buffer 1 (tested on at least 8 samples), which is typically used in standard 2D-GE, was followed by all precipitation protocols (i-v). With the TCA and TCA/acetone precipitations, the protein yield after precipitation was low (recovery 50% of input), the acetone precipitation tended to result in poor resolution of spots due to horizontal streaking, indicating that this precipitation method does not adequately remove contaminants that interfere with the focusing stage. In contrast, the 2D clean up kit consistently gave the best protein recovery and in conjunction with the SDS-based extraction buffer, appeared to provide the fullest representation of the proteome of the conditions tested. Murine epidermis is generally thinner than human epidermis and is of course furry. However, the ear skin has sparse hair follicles, and the transgenic pathological tissue has a massively thickened epidermis as well as dermis, in addition to the other tissues of the ear (such as cartilage). The protein extraction protocol proved to be very effective with this tissue (Figure 2) and is therefore likely to be perfectly suitable for human samples also. However, due to the relative insolubility of the cornified layer of the skin, it is possible that the SDS extraction buffer might lead to a quantitative underrepresentation of the components of this layer (although differentiation-specific keratins were readily detected), and if this was critical to the study, buffers 1 and 5 would provide a good urea-based alternative. Isoelectric focusing over two pH ranges, pH 4-7 and 6-9 was chosen and not pH 3-10 due to the poor resolution using the latter in the pH 4.5-6.0 range where the majority of proteins migrated. An alternative would be to use narrow pH range IPG strips covering a smaller pH range (known as zoom gels). This would obviously increase the number of experiments required to cover the full pH range, but instead, a single zoom gel at a critical pH (in this case 4.5-6.0) could be used in conjunction with the pH 3-10. An alternative approach to increase proteome coverage and simplify the spot pattern is by fractionation of the sample. Cells can be fractionated into distinct cellular compartments prior to protein extraction (reviewed in ref 19) or sequential extraction of protein fractions according to differences in solubility could be employed. Both would increase the number of DiGE experiments to be conducted. Once the DiGE experiments are run, several parameters must be considered. One is the criteria for difference; in this case, a base limit of 2-fold was taken (and
research articles only those of statistical significance). Given that we have found that the DiGE data generally underestimate the difference as detected by Western analysis (as described for SOD1 and keratin 6), it may be relevant to reduce (or even remove) the base limit, but maintain the criteria of statistical significance (or perhaps tighten this) to allow for biological variation. The choice of MS analysis will also affect the number of protein identifications obtained. Of the two techniques used here, MALDI-ToF is fast but has a lower identification success rate. Conversely, tandem MS has a better identification success rate but is more costly and time-consuming. There are a number of limitations associated with the DiGE approach specifically (such as the mechanism of protein labeling) and with 2D-GE comparisons in general. The CyDyes minimally label lysine residues; that is, the bulk of the protein sample is not labeled on lysine and therefore not visualized in the fluorescent images. This presents two problems. First, as the dyes have a molecular weight of ∼0.5 Da, the migration of labeled proteins through the gel is affected. Consequently, the position of a CyDye-labeled protein spot can differ slightly from the same unlabeled protein. This effect is more pronounced for lower molecular weight proteins and is the reason it is necessary to stain the bulk of the proteins for the preparative gel with a stoichiometric protein stain such as SYPRO orange. Such stains permit localization of the major portion of each protein species that has not been covalently labeled with CyDye. However, CyDye labeling may be more efficient in lysine-rich proteins. These factors can result in qualitative and quantitative differences in the spot maps that are obtained with protein stains and CyDye labels and matching the preparative gel to the analytical gels can thus be challenging. Including Cy2labeled pooled standard in the preparative gel facilitates this process by negating the requirement for gel-to-gel matching between CyDye-labeled analytical gels and the stained preparative gel. Nevertheless, we found that typically, 84% of spots could be reliably matched to the SYPRO orange image; that is, 16% of the differences could not be isolated or analyzed further. A general limitation of this type of approach reflects protein abundance and the difficulty in detecting proteins of low abundance, even if considerably differentially regulated. We have previously found several proteins of relatively low abundance to be differentially regulated in the transgenic tissue, such as p16 and MMP9, up-regulated in stage 5 tissue.4 These were not identified in the DiGE experiments conducted here. Detection of low-abundance proteins would be improved by the modifications discussed above, but it is not clear what the lower limits of this technique could be. An absolute limitation is in the identification of proteins, affected by the extraction of the protein from the gel plug, trypsinization as well as the spectrometry. In this study, 72% of the protein differences that were submitted for MS in one experiment were identified, while poorer gel resolution with the pH 6-9 experiment resulted in only 41% identification (Table 2). Published identification rates following DiGE vary from 41% to 69% with some reports indicating 100% identification of a small number of spots examined. In general, the identification rate for DiGE is lower than that for a whole proteome from standard 2D gels (which ranges between 80 and 95%) as DiGE, based on identification of differences, excludes many abundant, easily identified proteins. Despite the limitations, in these experiments, numerous differences were identified between transgenic pathological tissue and controls. Validation by Western blotting demonJournal of Proteome Research • Vol. 6, No. 9, 2007 3431
research articles strates that the results obtained are reliable. Two proteins identified by DiGE as differentially expressed, keratin 6 and SOD1, were examined and confirmed as up- and downregulated, respectively, by Western blotting. Interestingly, SOD1 showed differential regulation at both stages of the phenotype examined, but was only identified in the stage 5 pH 4-7 DiGE experiment. These data demonstrate that the DiGE system must not be considered as a comprehensive technique by which to identify all protein differences within a given sample. Even in circumstances where it is clear that the protein is extracted by the lysis buffer, can be represented on the 2D gel, and gives rise to a mass spectrum after digestion and extraction of peptides, identification of the difference is not always achieved. Many of the identified protein differences have plausible potential functional roles in the development of the pathology; for example, down-regulation of SOD1 may suggest that an imbalance in reactive oxygen species exists in the hyperplastic tissue, which could reflect the inflamed and degenerative phenotype. Similarly, keratin 6 induction (a marker of proliferation in the epidermis) was detected in the transgenic tissue, consistent with earlier results.3 In conclusion, the limitations and the methods required to optimize a DiGE comparison must be considered; nevertheless, the technique provides a reliable means to compare protein levels between samples and has been successfully used here for a complex tissue comprising skin, cartilage, and other components. The data provided by this analysis will be valuable in further exploring and understanding the basis for the transgene-induced pathology.
Acknowledgment. This work was supported by the Wellcome Trust (#069113/Z/02). We thank Andy Pitt for assistance with tandem MS, Kara McNair for technical advice, and David Greenhalgh for the anti-keratin antibodies. Proteomic analyses were performed in the Sir Henry Wellcome Functional Genomics Facility, University of Glasgow. Supporting Information Available: Supplementary Figures 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Unlu, M.; Morgan, M. E.; Minden, J. S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 1997, 18 (11), 2071-2077. (2) Marouga, R.; David, S.; Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal. Bioanal. Chem. 2005, 382 (3), 669-678. (3) Wilson, J. B.; Weinberg, W.; Johnson, R.; Yuspa, S.; Levine, A. J. Expression of the BNLF-1 oncogene of Epstein-Barr virus in the skin of transgenic mice induces hyperplasia and aberrant expression of keratin 6. Cell 1990, 61 (7), 1315-1327. (4) Stevenson, D.; Charalambous, C.; Wilson, J. B. Epstein-Barr Virus latent membrane protein 1 (CAO) up-regulates VEGF and TGFR concomitant with hyperlasia, with subsequent up-regulation of p16 and MMP9. Cancer Res. 2005, 65 (19), 8826-8835.
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Hannigan et al. (5) McNair, K.; Davies, C. H.; Cobb, S. R., Plasticity-related regulation of the hippocampal proteome. Eur. J. Neurosci. 2006, 23 (2), 575580. (6) Chromy, B. A.; Gonzales, A. D.; Perkins, J.; Choi, M. W.; Corzett, M. H.; Chang, B. C.; Corzett, C. H.; McCutchen-Maloney, S. L. Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins. J. Proteome Res. 2004, 3 (6), 1120-1127. (7) Yu, K. H.; Rustgi, A. K.; Blair, I. A. Characterization of proteins in human pancreatic cancer serum using differential gel electrophoresis and tandem mass spectrometry. J. Proteome Res. 2005, 4 (5), 1742-1751. (8) Lehr, S.; Kotzka, J.; Avci, H.; Knebel, B.; Muller, S.; Hanisch, F. G.; Jacob, S.; Haak, C.; Susanto, F.; Muller-Wieland, D. Effect of sterol regulatory element binding protein-1a on the mitochondrial protein pattern in human liver cells detected by 2D-DIGE. Biochemistry 2005, 44 (13), 5117-5128. (9) Leonard, J. F.; Courcol, M.; Mariet, C.; Charbonnier, A.; Boitier, E.; Duchesne, M.; Parker, F.; Genet, B.; Supatto, F.; Roberts, R.; Gautier, J. C. Proteomic characterization of the effects of clofibrate on protein expression in rat liver. Proteomics 2006, 6 (6), 19151933. (10) Zhou, G.; Li, H.; DeCamp, D.; Chen, S.; Shu, H.; Gong, Y.; Flaig, M.; Gillespie, J. W.; Hu, N.; Taylor, P. R.; Emmert-Buck, M. R.; Liotta, L. A.; Petricoin, E. F., III; Zhao, Y. 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol. Cell. Proteomics 2002, 1 (2), 117-124. (11) Human 2D-PAGE databases for proteome analyses in helath and disease home page, http://proteomics.cancer.dk. (12) Huang, C. M.; Foster, K. W.; DeSilva, T.; Zhang, J.; Shi, Z.; Yusuf, N.; Van Kampen, K. R.; Elmets, C. A.; Tang, D. C. Comparative proteomic profiling of murine skin. J. Invest. Dermatol. 2003, 121 (1), 51-64. (13) Huang, C. M.; Elmets, C. A.; van Kampen, K. R.; Desilva, T. S.; Barnes, S.; Kim, H.; Tang, D. C. Prospective highlights of functional skin proteomics. Mass Spectrom. Rev. 2005, 24 (5), 647-660. (14) Gromov, P.; Skovgaard, G. L.; Palsdottir, H.; Gromova, I.; Ostergaard, M.; Celis, J. E. Protein profiling of the human epidermis from the elderly reveals up-regulation of a signature of interferongamma-induced polypeptides that includes manganese-superoxide dismutase and the p85beta subunit of phosphatidylinositol 3-kinase. Mol. Cell. Proteomics 2003, 2 (2), 70-84. (15) Macdiarmid, J.; Stevenson, D.; Campbell, D. H.; Wilson, J. B. The latent membrane protein 1 of Epstein-Barr virus and loss of the INK4a locus: paradoxes resolve to cooperation in carcinogenesis in vivo. Carcinogenesis 2003, 24 (7), 1209-1218. (16) Rentrop, M.; Nischt, R.; Knapp, B.; Schweizer, J.; Winter, H. An unusual type-II 70-kilodalton keratin protein of mouse epidermis exhibiting postnatal body-site specificity and sensitivity to hyperproliferation. Differentiation 1987, 34 (3), 189-200. (17) Herzog, F.; Winter, H.; Schweizer, J. The large type II 70-kDa keratin of mouse epidermis is the ortholog of human keratin K2e. J. Invest. Dermatol. 1994, 102 (2), 165-170. (18) Matrix Science home page, http://www.matrixscience.com. (19) Yates, J. R., III; Gilchrist, A.; Howell, K. E.; Bergeron, J. J. Proteomics of organelles and large cellular structures. Nat. Rev. Mol. Cell. Biol. 2005, 6 (9), 702-714.
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