Comparative Proteomic Profiling of Murine Skin
2003; Elsevier BV; Volume: 121; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.2003.12327.x
ISSN1523-1747
AutoresChun‐Ming Huang, K. Wade Foster, Tivanka S. DeSilva, Jianfeng Zhang, Zhongkai Shi, Nabiha Yusuf, Kent R. Van Kampen, Craig A. Elmets, De-chu C. Tang,
Tópico(s)Bee Products Chemical Analysis
ResumoMammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes. Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes. adenine phosphoribosyltransferase two-dimensional gel electrophoresis protein disulfide isomerase 78 kDa glucose-regulated protein precursor heat shock protein isoelectric focusing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isoelectric point The skin is considered the largest organ of the human body and provides a physical and immunologic barrier to potentially harmful environmental agents (El Labban, 1982El Labban N.G. The nature of Langerhans cells granules: An ultrastructural study.Histopathology. 1982; 6: 317-325Crossref PubMed Scopus (13) Google Scholar;Segre et al., 1999Segre J.A. Bauer C. Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin.Nat Genet. 1999; 22: 356-360Crossref PubMed Scopus (587) Google Scholar). In addition to the ability to initiate an immune response against the antigens with which it comes in contact, the skin prohibits the entry of pathogens, regulates temperature, absorbs ultraviolet (UV) light, and prevents desiccation. Each of these environmental challenges causes changes in DNA conformation, alterations in the levels of mRNA and protein expression, and post-translational modifications of proteins specific to each stressor. Exposure of keratinocytes to UV radiation, for example, results in a cascade of molecular events including increases in the steady-state levels of mRNA encoding p21WAF1/CIP1 (Liu and Pelling, 1995Liu M. Pelling J.C. UV-B/A irradiation of mouse keratinocytes results in p53-mediated WAF1/CIP1 expression.Oncogene. 1995; 10: 1955-1960PubMed Google Scholar), increased levels of p53 (Qin et al., 2002Qin J.Z. Chaturvedi V. Denning M.F. Bacon P. Panella J. Choubey D. Nickoloff B.J. Regulation of apoptosis by p53 in UV-irradiated human epidermis, psoriatic plaques and senescent keratinocytes.Oncogene. 2002; 21: 2991-3002Crossref PubMed Scopus (85) Google Scholar), and changes in p53 phosphorylation (Chouinard et al., 2002Chouinard N. Valerie K. Rouabhia M. Huot J. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.Biochem J. 2002; 365: 133-145Crossref PubMed Scopus (102) Google Scholar). Various molecular approaches have contributed to a better understanding of how skin responds to the environment, and many important mediators of skin disease have been identified (Coulombe et al., 1991Coulombe P.A. Hutton M.E. Letai A. Hebert A. Paller A.S. Fuchs E. Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: Genetic and functional analyses.Cell. 1991; 66: 1301-1311Abstract Full Text PDF PubMed Scopus (507) Google Scholar;Guo et al., 1995Guo L. Degenstein L. Dowling J. Yu Q.C. Wollmann R. Perman B. Fuchs E. Gene targeting of BPAG1: Abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration.Cell. 1995; 81: 233-243Abstract Full Text PDF PubMed Scopus (387) Google Scholar;Johnson et al., 1996Johnson R.L. Rothman A.L. Xie J. et al.Human homolog of patched, a candidate gene for the basal cell nevus syndrome.Science. 1996; 272: 1668-1671Crossref PubMed Scopus (1571) Google Scholar). Despite the wealth of knowledge obtained from the use of molecular techniques, many critical cutaneous signaling pathways remain incompletely characterized or altogether unknown. New techniques have evolved to aid in the identification and characterization of large numbers of response elements involved in these pathways. One of these, oligonucleotide microarrays, produces a transcrip-tional profile of cells and can quantitatively analyze thousands of genes simultaneously, thereby allowing global characterization of a specific molecular process. Such technologic advances can be used to study the cutaneous response to environmental stressors. Recently several groups have employed this technique to better understand how the skin and cultured keratinocytes respond to injurious stimuli (Cole et al., 2001Cole J. Tsou R. Wallace K. Gibran N. Isik F. Early gene expression profile of human skin to injury using high-density cDNA microarrays.Wound Repair Regen. 2001; 9: 360-370Crossref PubMed Scopus (72) Google Scholar;Sesto et al., 2002Sesto A. Navarro M. Burslem F. Jorcano J.L. Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.Proc Natl Acad Sci USA. 2002; 99: 2965-2970Crossref PubMed Scopus (155) Google Scholar). Each study identified hundreds of responsive transcripts that were subsequently categorized into specific patterns of regulation. These efforts also identified novel responsive transcripts not previously associated with their respective processes. Although microarray technologies have enabled the analysis of large numbers of genes that respond to environmental stressors, correlation between mRNA and protein abundance is poor. Moreover, oligonucleotide microarrays cannot provide information concerning translational regulation of expression, post-translational modifications, or changes in steady-state levels of proteins. Proteomics provides a solution to this dilemma by profiling most, if not all, expressed proteins, their isoforms, post-translational modifications such as phosphorylation and glycosylation, and proteolytic cleavage. Proteomics is indispensable as there is no strict linear relationship between genes and the protein complement expressed within a cell, e.g., one transcript may produce a variety of protein products with diverse functions (Pandey and Mann, 2000Pandey A. Mann M. Proteomics to study genes and genomes.Nature. 2000; 405: 837-846Crossref PubMed Scopus (1850) Google Scholar). Conventional strategies for the study of protein function have focused on the analysis of single molecules. Although this stepwise approach has served biology well, large-scale proteomic initiatives have opened a new research vista in analysis of protein function. Although two-dimensional gel electrophoresis (2-DE) maps have been constructed from human keratinocytes and numerous murine tissue types (Celis et al., 1995Celis J.E. Rasmussen H.H. Gromov P. et al.The human keratinocyte two-dimensional gel protein database (update 1995): Mapping components of signal transduction pathways.Electrophoresis. 1995; 16: 2177-2240Crossref PubMed Scopus (109) Google Scholar;Sanchez et al., 2001Sanchez J.C. Chiappe D. Converset V. et al.The mouse SWISS-2D PAGE database: A tool for proteomics study of diabetes and obesity.Proteomics. 2001; 1: 136-163Crossref PubMed Scopus (135) Google Scholar), this study establishes a reference proteome map of whole skin, epidermis, and subepidermal tissues of BALB/c mice. From this map, we characterized 44 protein spots, corresponding to 28 different proteins, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, a computer searching algorithm, and an online protein database. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. The cutaneous proteins identified in this study have diverse functions, e.g., those involved in apoptosis and growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. These efforts also identified five molecular chaperones that exhibit differential responsiveness to temperature stresses. In addition to providing an important framework for future efforts aimed at characterizing cutaneous responses to environmental changes, this work provides a valuable starting point for the analysis of cultured and cocultured cells and the establishment of a comprehensive database containing qualitative and quantitative information about cutaneous proteins and their functions. BALB/c and C3H/HeN mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and Charles River Laboratories (Wilmington, MA), respectively. Animals were maintained in accordance with institutional guidelines. Abdominal skin was harvested from 2- to 3-mo-old female mice and subjected to 0.5 M ammonium thiocyanate at 37°C for 30 min to induce separation of the epidermis from subepidermal tissues when indicated (Bigby et al., 1987Bigby M. Kwan T. Sy M.S. Ratio of Langerhans cells to Thy-1+ dendritic epidermal cells in murine epidermis influences the intensity of contact hypersensitivity.J Invest Dermatol. 1987; 89: 495-499Abstract Full Text PDF PubMed Google Scholar). Epidermis, subepidermal tissues, or whole skin were then homogenized in lysis buffer containing 9.5 M urea, 4% CHAPS, 5% tributylphosphine, 1.6% pH 5–8 Bio-lytes, 0.4% pH 3–10 Bio-Lytes, and Complete Protease Inhibitor Cocktail (Cat. no. 1697498, Roche, Mannheim, Germany). Tissue homogenates were centrifuged at 2,000 g for 10 min at room temperature and soluble protein was quantitated as described previously (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (205559) Google Scholar;Huang et al., 2001Huang C.M. Shui H.A. Wu Y.T. Chu P.W. Lin K.G. Kao L.S. Chen S.T. Proteomic analysis of proteins in PC12 cells before and after treatment with nerve growth factor: Increased levels of a 43-kDa chromogranin B-derived fragment during neuronal differentiation.Brain Res Mol Brain Res. 2001; 92: 181-192Crossref PubMed Scopus (26) Google Scholar). Chaperone expression in murine ventral skin was analyzed following exposure to temperature stress. All mice were anesthetized using 10 mg ketamine and 1.5 mg xylazine per 100 g body weight. Ventral hair was removed using a Norelco T900 electric trimmer, and mice were situated such that ventral skin was in direct contact with ice at 0°C, the plastic cage bottom at 25°C (control), or a pre-equilibrated glass culture dish immersed in a 45°C water bath. Ventral skin was exposed to elevated, reduced, or control temperatures for 10 min, immediately harvested, and then homogenized in lysis buffer. Subsequent sample preparations were conducted as described above. Aliquots containing 300 μg protein were mixed 1:1 with rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 2% SB 3–10, 5 mM tributylphosphine, 1.6% pH 5–8 Bio-lytes, 0.4% pH 3–10 Bio-Lytes, and trace bromophenol blue, as described previously (Rabilloud, 2002Rabilloud T. Two-dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still climbs up the mountains.Proteomics. 2002; 2: 3-10Crossref PubMed Scopus (657) Google Scholar). Samples were subjected to isoelectric focusing (IEF) in 13 cm linear gradient Immobiline Dry-Strips, pH 3–10 or pH 4–7, for 60 kV h using a Pharmacia Hoefer Multiphor II electrophoresis chamber. Following IEF, Dry-Strips were incubated at room temperature for 20 min in equilibration solution containing 50 mM Tris–HCl, pH 8.8, 6 M urea, 2% sodium dodecyl sulfate (SDS), 30% glycerol, and 5 mM tributylphosphine. Dry-Strips were then embedded in 1% agarose containing trace bromophenol blue and loaded onto a large format (12.5 cm×20 cm), 8%–16% gradient SDS polyacrylamide gel. Electrophoresis was conducted at 200 V for 5–6 h or 30 mA per gel overnight until the bromophenol blue dye front was within 2 cm of the bottom of the gel. Polyacrylamide gels were then stained with Coomassie blue or silver nitrate as described previously (Shevchenko et al., 1996Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal Chem. 1996; 68: 850-858Crossref PubMed Scopus (7579) Google Scholar). Silver-stained gels were scanned using a Molecular Dynamics Personal Densitometer, and protein spots were quantified and matched using PDQuest software (Bio-Rad, Hercules, CA). Each two-dimensional gel from BALB/c or C3H/HeN mouse skin is representative of experiments conducted in triplicate from separate skin harvests. Identical results were obtained in each of three two-dimensional gels run on either BALB/c- or C3H/HeN-derived extracts. The localization and number of spots were identical in each run from the same or separate harvests of BALB/c or C3H/HeN mouse skin. All differences in gel spot density between groups were verified manually to rule out the possibility of artifacts. To address variability in silver-staining, individual gel spot volumes were normalized by dividing their optical density values by the total optical density values of all the spots present in the gel. Differences in protein expression between epidermis and subepidermal tissues were compared using Student's t test. In-gel digestion was performed essentially as described previously (Kaji et al., 2000Kaji H. Tsuji T. Mawuenyega K.G. Wakamiya A. Taoka M. Isobe T. Profiling of Caenorhabditis elegans proteins using two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time of flight mass spectrometry.Electrophoresis. 2000; 21: 1755-1765Crossref PubMed Scopus (49) Google Scholar). Protein spots excised from the Coomassie-blue- or silver-stained gel were destained in 0.2 ml acetonitrile for 15 min and dried to completion in a SpeedVac vacuum centrifuge. Samples were then rehydrated on ice for 45 min in digestion buffer (50 mM acetonitrile, 0.04 mg per ml modified trypsin; Promega, Madison, WI). After removing excess solution, proteins were further digested at 37°C for 15 h. The resultant peptides were extracted with 5% formic acid in 50% acetonitrile and desalted and concentrated using ZipTips containing C18 resin (Millipore, Bedford, MA). Peptides were eluted from the ZipTips with 75% acetonitrile/0.1% trifluoroacetic acid, applied to the sample target, and air-dried. Peptide fragments were then reconstituted in matrix solution containing α-cyano-4-hydroxycinnamic acid dissolved in 50% acetonitrile/0.1% trifluoroacetic acid and analyzed with a PerSeptive Voyager-DE MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA). Peptides were evaporated with an N2 laser at 337 nm. Each spectrum was the cumulative average of 50–100 laser shots. All peptide samples were measured as mono-isotopic masses, and autolytic peaks of trypsin were used for internal calibration. Up to one missed trypsin cleavage was allowed, although most matches did not contain any missed cleavages. This procedure resulted in mass accuracies of 100 ppm. Peptide fingerprint mass spectra exceeding 5% of full scale were analyzed, interpreted, and matched to SWISS-PROT database entries using Mascot, a searching algorithm available at the Matrix Science Homepage, http://www.matrixscience.com. Matches were computed using a probability-based Mowse score defined as –10 × log p, where p is the probability that the observed match was a random event (Perkins et al., 1999Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6485) Google Scholar). Mowse scores greater than 70 were considered significant (p≤0.05). BALB/c ventral skin was subjected to 0.5 M ammonium thiocyanate at 37°C for 30 min resulting in separation of epidermal and subepidermal tissues. Total RNA was isolated using TRIzol Reagent (Invitrogen, Life Technologies, Carlsbad, CA). Nested RT-PCR was performed using the following primer sets for prohibitin and adenine phosphoribosyltransferase (APRT). External prohibitin primer sets were 5′-GGA GTC ATG GCT GCC AAA GTG TTT GAG-3′ and 5′-GGT GAT GTT CCG AGA GCG GGA GAG CTG-3′ resulting in amplification of a 783 bp fragment. Nested (internal) prohibitin primers were 5′-GCA GTT GCA GGA GGC GTG GTG AAC TCT-3′ and 5′-CTT TTC CAC CAC AAA TCT GGC TCT CTC-3′ resulting in amplification of an internal 558 bp subfragment. Amplification of APRT was used as an internal control. External APRT primers were 5′-GCC AGT CAC CTG AAG TCC ACG CAC AGC-3′ and 5′-TCA GTC ATA CTG GAG GAG AGA GAA GAA-3′ resulting in amplification of a 395 bp fragment. Nested (internal) APRT primers were 5′-CTG TGT GCT CAT CCG GAA ACA GGG GAA-3′ and 5′-AGC AGG TCA CAG GCC GCA AAC ATG GTT-3′ resulting in amplification of a 173 bp subfragment. Following initial amplification using external primer pairs, PCR products were diluted in 10-fold increments and subjected to 35 cycles of nested PCR amplification using internal primer sets. Amplified DNA fragments were separated on a 1.5% agarose gel. Five micron cryosections were cut from BALB/c female ventral skin and fixed in 3% formaldehyde in phosphate-buffered saline (PBS) for 45 min at room temperature. Following fixation, sections were permeabilized with 0.5% Triton X-100 in PBS for 2 min and blocked with 1% bovine serum albumin in PBS for 30 min. Tissues were then incubated with primary antibody in dilution buffer containing 1% bovine serum albumin and 0.01% Tween 20 in PBS for 45 min at 37°C. Following incubation with primary antibody, tissues were washed three times for 10 min each in PBS at 4C and subsequently incubated with 1% bovine serum albumin in PBS at 4°C for 20 min. Tissues incubated with anti heat shock protein 27 (anti-HSP27) primary antibody were subsequently treated with Alexa Fluor 488 conjugated donkey antigoat IgG, and tissues incubated with anti-keratin 10 (anti-K10) or anti-K14 primary antibodies were subsequently treated with Alexa Fluor 594 goat antirabbit IgG. Major histocompatibility complex (MHC) class II and CD3 antigens were detected by incubation of tissue sections with the respective biotinylated primary antibody at 0.5 mg per ml in dilution buffer for 45 min at 37°C. Tissues were then washed three times for 10 min in PBS at 4°C, incubated with 1% bovine serum albumin in PBS at 4°C for 20 min, and subsequently treated with R-phycoerythrin-conjugated streptavidin diluted 1:500 in dilution buffer for 45 min at 37°C in the dark. Following incubation with secondary antibody or R-phycoerythrin-conjugated streptavidin, tissue sections were washed for 10 min each in PBS, counterstained with 20 μg per ml Hoechst 33258 for 4 min, and mounted in polyvinyl alcohol mounting medium containing 10% (wt/vol) polyvinyl alcohol, 25% (vol/vol) glycerol, and 50 mM Tris–HCl, pH 8.5. Fluorescent emissions were detected with a Zeiss Axiovert 100 microscope with epifluorescent optics and analyzed with LSM 3.8 software (Carl Zeiss, Oberkochen, Germany). Dilution factors for anti-HSP27, anti-K10, and anti-K14 antibodies were 1:100, 1:500, and 1:1000, respectively. Goat polyclonal anti-HSP27 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-K10 and anti-K14 antibodies were from Berkeley Antibody (Richmond, CA). R-phycoerythrin-conjugated streptavidin, biotinylated anti-MHC II, anti-CD3 primary antibodies, Alexa Fluor 488 conjugated donkey antigoat IgG, and Alexa Fluor 594 conjugated goat antirabbit IgG were from Molecular Probes (Eugene, OR). Aliquots containing 300 μg protein from BALB/c skin were separated in a 12.5% SDS polyacrylamide gel and then electrophoretically transferred (1.5 h at 24 V) to PVDF membranes for blotting. HSP27-, HSP60-, HSP70- and actin-specific antibodies (Santa Cruz Biotechnology) were diluted 1:500. Proteins were visualized by chemiluminescence reaction. Proteome reference maps of BALB/c murine abdominal skin were constructed by IEF within linear pH gradients ranging from 3 to 10 (Figure 1a) and from 4 to 7 (Figure 1b) followed by SDS polyacrylamide gel electrophoresis and silver-staining. Using PDQuest software, a total of 526 protein spots were resolved by IEF within linear gradient pH 4–7. The localization and number of spots were identical in triplicate gels from independent skin harvests. Of the 526 protein spots, 44 have thus far been identified using MALDI-TOF MS and a probability-based database searching algorithm (Table I). In general, there was good correlation between the observed and theoretical isoelectric point (pI) and Mr values of the 44 protein spots identified. Detectable differences between the observed and theoretical Mr (spots 1, 2, 5, 6, 7, 15, 16, 29, 30, 35, 44–47, 52) may be secondary to post-translational modifications or the presence of protein isoforms, as exemplified by α-actin and myosin in a similar experiment (Verrills et al., 2000Verrills N.M. Harry J.H. Walsh B.J. Hains P.G. Robinson E.S. Cross-matching marsupial proteins with eutherian mammal databases: Proteome analysis of cells from UV-induced skin tumours of an opossum (Monodelphis domestica).Electrophoresis. 2000; 21: 3810-3822Crossref PubMed Scopus (12) Google Scholar). Observed and theoretical pI values were also different in certain cases. The observed pI values of annexin I (spot 44), β-tropomyosin (spot 45), and creatine kinase, M chain (spot 51), for example, were 5.90, 5.81, and 7.32, respectively, and differed from theoretical pI values, 7.15, 4.66, and 6.58, respectively.Table IProteins identified from the proteome map of BALB/c skinaTheoretical (Theor.) pI and Mr values were calculated using the ExPASY Compute pI/Mw tool at http://us.expasy.org/tools/pi-tool.html. Observed (Obs.) pI and Mr values were calculated with PDQuest software (Bio-Rad). Confirmation of protein expression and/or correlation of protein expression with gene expression were undertaken wherever possible: HSP27 (Wakayama and Iseki, 1998); vimentin (Tyner et al, 1985); K10 (Roop et al, 1984); K14 (Coulombe et al, 1989); K15 (Waseem et al, 1999); Cu/Zn superoxide dismutase (Sander et al, 2002); galectin-7 (Magnaldo et al, 1998); parvalbumin α (Berchtold and Means, 1985); procollagen, type VI, α1 (Olsen et al, 1989); HSP70 (Huang et al, 2001); HSP60 (Laplante et al, 1998); annexin I (Sato-Matsumura et al, 1996); annexin II (Karimi-Busheri et al, 2002). Expression of prohibitin and HSP27 was verified by RT-PCR and immunohistochemistry, respectively.Mr (kDa)pISpotProtein/FunctionGeneTheor.Obs.Theor.Obs.SWISS-PROT Accession No.Observed m/z and predicted peptide sequencebThe predicted amino acid sequence for signature m/z fragments is given in parentheses.Sequence coverage (%)Epidermis rich1HSP27/HSP2723.0126.806.126.10P146021075.60 (84–93)22Molecular chaperone1104.51 (132–140)1149.63 (29-38)1832.99 (176–192)2HSP27/HSP2723.0127.006.125.80P146021104.51 (132–140)17Molecular chaperone1149.63 (29-38)1832.99 (176–192)3Vimentin/VIM53.5653.435.065.06P201521125.61 (114–122)20Intermediate filament1308.65 (293–304)1539.87 (130–143)1557.89 (411–424)1587.80 (101–113)1838.90 (425–440)2126.07 (79–97)4Vimentin/VIM53.5653.435.065.04P201521088.57(208–217)16Intermediate filament1093.54 (295–304)1121.54 (382–390)1125.60 (114–122)1433.69 (322–334)1444.71 (51–64)1557.92 (411–424)5K10/KRT1057.7153.265.015.07P025351165.64 (440–448)15Intermediate filament1167.58 (183–192)1201.63 (244–254)1264.72 (234–243)1357.73 (244–255)1390.70 (385–397)1406.65 (449–464)1422.76 (438–448)1769.97 (369–384)6K10/KRT1057.7153.265.015.05P025351165.64 (440–448)7Intermediate filament1167.58 (183–192)1264.72 (234–243)1357.73 (244–255)1422.76 (438–448)7K14/KRT1441.4849.214.915.14Q617811167.65 (347–356)13Intermediate filament1345.68 (16–27)1429.57 (270–280)1892.94 (218–233)8K15/KRT1549.1648.214.784.78Q61414989.48 (145–151)18Intermediate filament1028.62 (170–178)1029.62 (208–216)1064.59 (109–117)1073.59 (160–169)1122.63 (392–400)1278.70 (186–195)1350.60 (273–283)1729.96 (401–416)9K15/KRT1549.1647.914.784.78Q614141028.60 (170–178)17Intermediate filament1029.60 (208–216)1073.59 (160–169)1122.63 (392–400)1278.70 (186–195)1327.79 (196–207)1500.70 (24–41)10α-Actin/ACTC142.0243.005.235.04Q9CXK3923.51 (329–336)14Microfilament976.50 (19–28)1130.60 (197–206)1198.74 (29–39)1790.92 (239–254)11α-Actin/ACTC142.0241.105.235.05Q9CXK3976.50 (19–28)12Microfilament1130.60 (197–206)1198.74 (29–39)1790.92 (239–254)12α-Actin/ACTC142.0240.315.235.05Q9CXK3923.51 (329–336)12Microfilament1130.60 (197–206)1198.74 (29–39)1790.92 (239–254)13α-Actin/ACTC142.0243.005.235.40Q9CXK3923.51 (329–336)12Microfilament1130.60 (197–206)1198.74 (29–39)1790.92 (239–254)14α-Actin/ACTC142.0242.165.235.56Q9CXK3923.57 (9329–336)19Microfilament1130.57 (197–206)1500.75 (360–372)1790.91 (239–254)1956.02 (96–113)15α-Actin/ACTC142.0239.165.235.56Q9CXK3976.50 (19–28)12Microfilament1130.60 (197–206)1198.74 (29–39)1790.92 (239–254)1640S ribosomal protein SA/LAMR132.7243.004.744.72P142061135.64 (43–52)17Transcriptional control1203.65 (90–102)1614.80 (167–180)1698.86 (103–117)17Prohibitin/PHB29.8229.805.575.56P241421058.47 (187–195)26Growth inhibitor1149.52 (134–143)1185.60 (84–93)1396.77 (94–105)1460.61 (106–117)1998.06 (220–229)18Cu/Zn superoxide dismutase/SOD115.8115.836.036.03P08228967.36 (72–80)30Scavenger of free radicals1095.46 (71–80)1367.72 (104–116)2323.22 (81–103)19Galectin-7/LGALS715.0415.046.697.00O549741252.58 (55–65)38Apoptotic response1290.60 (73–83)1479.77 (121–134)1694.79 (100–113)1777.90 (84–99)20–25NIcNI, proteins not identified.––––––––Subepidermis rich26ER60/ERP6056.6256.635.995.98P277731191.57 (63–73)17Molecular chaperone1258.66 (183–193)1341.68 (448–459)1397.77 (471–481)1652.78 (104–118)1587.87 (147–160)1758.93 (130–145)27Myosin light chain 1/MYL120.4620.464.985.04P059771010.54 (51–58)27Microfilament1200.70 (64–74)1484.66 (119–131)1819.03 (9–28)28Myosin light chain 1/MYL120.4620.464.985.06P059771010.54 (51–58)27Microfilament1200.70 (64–74)1484.66 (119–131)1819.03 (9–28)29Myosin light chain 1/MYL120.4618.234.984.72P059771010.54 (51–58)27Microfilament1200.70 (64–74)1484.66 (119–131)1819.03 (9–28)30H+-transporting ATP synthase/ATP5B54.4349.214.984.98P56480975.56 (202–212)8Energy metabolism1038.59 (134–143)1262.64 (110–121)1457.84 (213–225)31Parvalbumin α/PVA11.8011.825.024.72P328481260.66 (85–97)39Calcium binding1420.71 (15–28)1536.79 (56–69)32α-Enolase/EN0147.0148.026.366.40P171
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