Protein Database, Human Retinal Pigment Epithelium
2003; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês
10.1074/mcp.d200001-mcp200
ISSN1535-9484
AutoresKaren A. West, Lin Yan, K.G. Shadrach, Sun Jian, Azeem Hasan, Masaru Miyagi, John S. Crabb, Joe G. Hollyfield, Alan D. Marmorstein, John W. Crabb,
Tópico(s)Retinal Imaging and Analysis
ResumoThe retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome. The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome. The RPE 1The abbreviations used are: RPE, retinal pigment epithelium; 1D, one-dimensional; 2D, two-dimensional; IEF, isoelectric focusing; IPG, immobilized pH gradient; LC MS/MS, liquid chromatography tandem electrospray mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is a simple cuboidal epithelium that separates the photoreceptor cells of the retina from their principal blood supply in the choroid (1.Marmor M.F. Wolfensberger T.J. The Retinal Pigment Epithelium, Function and Disease, Oxford University Press, New York1998Google Scholar). In all vertebrates, the RPE forms an integral part of the blood-retinal barrier and is responsible for vectorial transport of nutrients to rod and cone photoreceptors and removal of waste products to the blood. In addition, the RPE phagocytizes shed photoreceptor outer segments, absorbs scattered light, and functions in the retinoid visual cycle and regeneration of bleached visual pigment (2.Bok D. The retinal pigment epithelium: a versatile partner in vision.J. Cell Sci. Suppl. 1993; 17: 189-195Google Scholar, 3.Marmorstein A.D. The polarity of the retinal pigment epithelium.Traffic. 2001; 2: 867-872Google Scholar). To facilitate studies of retina and RPE in health and disease (4.Crabb J.W. Miyagi M. Gu X. Shadrach K. West K.A. Sakaguchi H. Kamei M. Hasan A. Yan L. Rayborn M.E. Salomon R.G. Hollyfield J.G. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14682-14687Google Scholar), we have initiated the development of a human RPE protein database. Forty-two normal human eyes were used in this study and were obtained from the Cleveland Eye Bank within 3–12 h postmortem. Following bisection of the globe behind the limbus, the anterior segment and vitreous were discarded, and the retina was removed. RPE cells were gently brushed from the eye cup using an artist's 7-mm angular paint brush (Langnickel L7160) and Ca2+- and Mg2+-free phosphate-buffered saline containing 5 mm EDTA and 1 mm phenylmethylsulfonyl fluoride (two to three times with 500 μl). The RPE cells were then washed two to three times by centrifugation in phosphate-buffered saline. For select preparations, red blood cells were removed by centrifugation in a Percoll gradient (1.0–1.1 g/ml, Amersham Biosciences); the pigmented RPE cells float near the top of the gradient and were collected with a Pasteur pipette and then washed two times in phosphate-buffered saline. Whole cell lysates were prepared by homogenizing RPE cell pellets in isoelectric focusing (IEF) solvent B (7 m urea, 2 m thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholyte, 1% dithiothreitol), the solution was clarified by centrifugation, and protein was quantified by a modified Bradford assay (5.Ramagli L.S. Link A.J. Methods in Molecular Biology, 2D-Proteome Analysis Protocols. 112. Humana Press, Totawa, NJ1999: 99-103Google Scholar). About 95 μg of soluble whole cell RPE protein was recovered per eye (n = 11 eyes). Subcellular RPE fractions were prepared according to Saari et al. (6.Saari J.C. Bredberg D.L. CoA- and non-CoA-dependent retinol esterification in retinal pigment epithelium.J. Biol. Chem. 1988; 263: 8040-8090Google Scholar). Briefly, freshly isolated RPE cells were suspended in 1–4 ml of 25 mm Tris acetate, pH 7, 0.25 m sucrose, 1 mm dithiothreitol, homogenized with 25–125 manual passes of a glass homogenizer, and clarified in a microcentrifuge for 10 min at 1,000 × g. The clarified RPE lysate was centrifuged at 27,000 × g at 5 °C for 20 min, yielding the P2 membrane fraction. The supernatant was centrifuged again at 150,000 × g for 1 h at 5 °C, yielding the microsomal and cytosolic cell fractions. The microsomal and P2 pellets were resuspended in IEF solvent B. The cytosolic fraction was exchanged into IEF solvent B using Centricon concentrators (Amicon, 10-kDa molecular mass cut-off). Average recovery per eye was about 18 μg of cytosolic protein, 16 μg of P2 membrane protein, and 9 μg of microsomal protein (n = 31 eyes) based on the modified Bradford assay (5.Ramagli L.S. Link A.J. Methods in Molecular Biology, 2D-Proteome Analysis Protocols. 112. Humana Press, Totawa, NJ1999: 99-103Google Scholar). One- and two-dimensional electrophoresis was performed as described previously using the Bio-Rad Mini-Protein II, Bio-Rad Protein IIxi, Amersham Biosciences IPGphor, and Amersham Biosciences IsoDalt systems (7.West K.A. Yan L. Miyagi M. Crabb J.S. Marmorstein A.D. Marmorstein L. Crabb J.W. Proteome survey of proliferating and differentiating rat retinal pigment epithelial J-cells.Exp. Eye. Res. 2001; 73: 479-491Google Scholar, 8.Aulak K.S. Miyagi M. Yan L. West K.A. Massillon D. Crabb J.W. Stuehr D.J. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12056-12061Google Scholar, 9.Miyagi M. Sakaguchi H. Darrow R.M. Yan L. West K.A. Aulak K.S. Stuehr D.J. Hollyfield J.G. Organisciak D.T. Crabb J.W. Evidence that light modulates protein nitration in rat retina.Mol. Cell. Proteomics. 2002; 1: 293-303Google Scholar). Isoelectric focusing was performed with non-linear pH 3–10 or linear pH 4–7 immobilized pH gradients (18-cm IPG strips, Amersham Biosciences) in 7 m urea, 2 m thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholytes, 1% dithiothreitol. Second dimension electrophoresis utilized 23.5- × 18- × 0.1-cm gels (12% acrylamide). Colloidal Coomassie Blue- (Pierce Code Blue) or silver-stained gel patterns were recorded with Quantity One and PDQuest gel analysis software (Bio-Rad). Protein from multiple eyes was utilized for most electrophoretic separations, and amounts varied from ∼30 to ∼500 μg/gel. Identification of proteins by peptide mass mapping and/or liquid chromatography electrospray tandem mass spectrometry (LC MS/MS) were as described elsewhere (7.West K.A. Yan L. Miyagi M. Crabb J.S. Marmorstein A.D. Marmorstein L. Crabb J.W. Proteome survey of proliferating and differentiating rat retinal pigment epithelial J-cells.Exp. Eye. Res. 2001; 73: 479-491Google Scholar, 8.Aulak K.S. Miyagi M. Yan L. West K.A. Massillon D. Crabb J.W. Stuehr D.J. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12056-12061Google Scholar, 9.Miyagi M. Sakaguchi H. Darrow R.M. Yan L. West K.A. Aulak K.S. Stuehr D.J. Hollyfield J.G. Organisciak D.T. Crabb J.W. Evidence that light modulates protein nitration in rat retina.Mol. Cell. Proteomics. 2002; 1: 293-303Google Scholar). Briefly, gel spots and bands were excised, stain was washed away, proteins were digested in-gel with trypsin, and peptides were extracted for mass spectrometric analysis. For MALDI-TOF MS, peptides were adsorbed onto C18 ZipTips (Millipore, Bedford, MA), eluted with 75% acetonitrile, 0.02% trifluoroacetic acid, and analyzed using a Voyager DE Pro MALDI-TOF mass spectrometer (PE Biosystems, Framingham, MA). Measured peptide masses were used to query the Swiss Protein, TrEMBL, and National Center for Biotechnology Information (NCBI) sequence databases for matches using MS-Fit and Profound search programs and a mass tolerance of 50 ppm. Positive identification by peptide mass mapping required four to five peptide matches under the search conditions used (10.Jensen O.N. Wilm M. Schevchenko A. Mann M. Link A.J. Methods in Molecular Biology, 2D-Proteome Analysis Protocols. 112. Humana Press, Totawa, NJ1999: 513-530Google Scholar). For analysis by LC MS/MS, tryptic digests were injected by autosampler onto a 0.3- × 1-mm trapping column (PepMap C18, LC Packings) using a CapLC system (Micromass, Beverly, MA). Peptides were eluted at 250 nl/min and chromatographed on Biobasic C18 columns (50 μm × 5 cm or 75 μm × 5 cm, New Objective, Cambridge, MA) directly into a quadrupole time-of-flight mass spectrometer (QTOF2, Micromass). Protein identifications from MS/MS data utilized Micromass software ProteinLynx™ Global Server, MassLynx™, version 3.5, and the Swiss Protein and NCBI protein sequence databases. MS/MS spectra were examined manually to verify determined sequences. Two hundred seventy-eight proteins were identified in proteomic analyses of the human RPE and are listed in Table I with database accession numbers, cell subfraction of origin, and summaries of electrophoretic and mass spectrometric data supporting the identifications. A total of 17 2D gels were performed with RPE cell fractions, including eight for method development and nine in which spots were excised and analyzed (five for whole cell, three for cytosolic, three for microsomal, and one for P2 cell fractions). Representative 2D gels for each cell fraction are shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4. In addition, bands were excised and analyzed from two 1D SDS-PAGE separations of the P2, microsomal, and cytosolic RPE cell fractions (Fig. 5). One hundred sixty proteins were identified following 2D PAGE, 180 proteins were identified following 1D PAGE, and 62 proteins were identified from both 1D and 2D gels. The 1D and 2D PAGE methods were complimentary, each contributing unique protein identifications to this preliminary proteome analysis. Other profiling of human RPE gene expression has been obtained through serial analysis of gene expression (SAGE), which yielded the identity of 445 genes (227 with assigned functions) and 333 unknown sequence tags (11.Sharon D. Blackshaw S. Cepko C.L. Dryja T.P. Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE).Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 315-320Google Scholar). Expressed sequence tag analysis of combined human RPE and choroid tissue has also been reported (12.Wistow G. Bernstein S.L. Wyatt M.K. Farris R.N. Behal A. Touchman J.W. Bouffard G. Smith D. Peterson K. Expressed sequence tag analysis of human RPE/choroid for the NEIBank project: over 6000 non-redundant transcripts, novel genes and splice variants.Mol. Vis. 2002; 8: 205-220Google Scholar); however, the identify of RPE components was obscured by the choroid vasculature. Less than half (44%) of the proteins recently identified from in vitro cultures of rat RPE-J cells (7.West K.A. Yan L. Miyagi M. Crabb J.S. Marmorstein A.D. Marmorstein L. Crabb J.W. Proteome survey of proliferating and differentiating rat retinal pigment epithelial J-cells.Exp. Eye. Res. 2001; 73: 479-491Google Scholar) were also observed in the present analysis of human in vivo RPE.Table IProteins identified in human retinal pigmentProteinAccession number aSwiss Protein database accession numbers are shown in plain font. Links to Swiss Protein accession numbers use the EXPASY server at http://us.expasy.org/sprot/. NCBI accession numbers are in italics. Links to NCBI accession numbers use the Entrez server at http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Protein. Parenthesis following accession numbers designate identifications based on homology with other species: A, Anopheles albimanus; B, Buchuera aphyidicola; C, Carassius auratus; D, Dictyostelium discoideum; F, Fragaria x ananassu; H, Helicobacter pylori; J, Bacillus; L, Amaranthus hypochondriacus; M, Mus musculus; N, Methanocaldococcus jannaschii; Q, Borrelia burgdorferi; R, Rattus norvegicus; T, Hevea brasiliensis; V, Volvox carter; X, Xenopus laevis.MALDI-TOF MSLC MS/MSCell fraction dCell fractions in which the protein was found: WC, whole cell; C, cytosolic; P, P2 membrane; M, microsomal.1D gel cell fraction/∼molecular mass (kDa) eSDS-PAGE are shown for C, P2, and M cell fractions in electronic Fig. 5. Identified proteins were excised from the indicated molecular mass region.2D gel cell fraction/spot no. fRepresentative 2D PAGE profiles for WC, C, P2, and M cell fractions are shown in electronic Figs. 1–4, respectively. Identified proteins were excised from the numbered spots in the figures and/or from the indicated molecular mass and pI range in other 2D gels.Molecular mass (kDa)/pIMatches (error ppm) bAverage error of measured peptide masses is shown for identifications based solely on peptide mass mapping.Coverage %MatchesPeptides identified cPeptide sequences are shown for identifications based on a single peptide (underlined residues were determined).2D gel observedCalculatedAcetyl-CoA acetyltransferase 1P247525197C, WCC 40C 945/7.345.20/8.98Acetyl-CoA acyltransferase 2P427654CC 43C 540/5.341.91/8.50Acid ceramidaseQ135103CC 3844.64/7.52Acidic ribosomal 60 S protein P0P053886 (18)28MM 2040/5.834.27/5.72Acidic ribosomal 60 S protein P2P053873WC11/4.511.66/4.42Aconitate hydrataseQ99798112116C, WCC 97WC 22, C 2194/6.985.42/7.36Actin 1, αP0256883410PP 43P 445/5.241.83/5.30Actin, βP025709356M, C, WCM 13, C 2644/5.1–5.3, 41.5/5.341.74/5.29Actin, γP025718344C, P, WCC 43P 3, P 6, WC 3084/5.141.60/5.45Acyl-CoA dehydrogenase, C2 to C3 short chainP162192CC 3944.30/8.13Acyl-CoA dehydrogenase, medium chain-specificP113104CC 40, C 4346.58/8.61Acyl-CoA dehydrogenase, very long chain-specificP497487CC 7070.39/8.92Acyl-CoA oxidase 1Q150672CC 5074.44/8.35Adenylate kinase isoenzyme 1P005685 (21)31CC 1027/7.821.67/8.40ADP, ATP carrier proteinP051415PP 3332.83/9.84Aflatoxin B1 aldehyde reductaseO434881FFGNSWAETYRCC 3633.06/9.78Aldehyde dehydrogenase, E2 isozymeP0509112CC 5556.38/6.63Aldehyde dehydrogenase, E3 isozymeP491896M, CC 5453.53/6.01Aldolase A, fructose-bisphosphateP0407510 (7)29WCWC 1142/7.539.28/8.39Aldolase C, fructose-bisphosphateP0997210402M, C, WCC 35WC 19, M 46, C 1943/6.5, 43/6.9, 43/6.639.44/6.41α1-AntichymotrypsinP010113CC 3465/4.425.18/5.64α1-AntitrypsinP0100911336C, WCWC 465/5.046.73/5.37AMBP protein precursorP027601MTVSTLVLGEGATEAEISMTSTRWC41/7.138.98/6.20Aminobutyrate aminotransferaseP804042CC 5356.55/7.56Annexin IIP073555C38/7.438.45/8.00Annexin IVP095251GLGTDEDAIISVLAYRCC 2235.75/5.85Annexin VP087584CC 2235.94/4.94Annexin VIP081334CC 7575.74/5.42Antioxidant protein 2P300413WC46/6.424.89/6.30AntiquitinP494195MC 5560/6.555.36/6.44Apolipoprotein A-IP0264711447MM 1230/5.230.77/5.56Arrestin CP365756 (13)21MM 4151/5.542.76/5.53Aspartate aminotransferaseP0050592013C, WCC 97191.61/5.48ClusterinP109091EILSVDCSTNNPSQAKWC38/5.452.48/6.20c-Myc promotor-binding proteinP227122CC 2036/5.637.07/7.20Cofilin non-muscleP235283CC 3022/7.418.54/8.50Complement C1Q070213CC 21, C 3331.36/4.74Complement C3P010241VFSLAVNLIAIDSQVLCGAVKWC44/6.5187.15/6.30Complement C9P027481GTVIDVTDFVNWASSINDAPVLISQKWC44/6.4, 56/5.863.16/5.50Creatine kinase A1QK1A1GTGGVDTAAVGGVFDVSNADRWC45/5.042.72/6.30Creatine kinase ubiquitous mitochondrialP125325MM 4845/7.547.02/8.7Creatine kinase, B chainP1227718506M, C, WCM 43WC 28, M 48, C 2547/5.3, 46/7.4, 44/5.342.64/5.34Creatine kinase, M chainP067321LEKGQSIDDMIPAQKCC 2540/5.543.08/7.20Crystallin A chain, αP024891FVIFLDVKMM 2519.74/5.78Crystallin B chain, αP025116 (7)30P, WCWC 1627/6.620.16/6.76Crystallin ζQ082575CC 3535.21/8.56Cytochrome b5P001675 (11)69WC13/5.011.27/5.02Cytochrome c oxidase polypeptide VbP106061GLDPYNVLAPKPP 1911.65/9.9Δ1-Pyrroline-5-carboxylate dehydrogenaseP300389225C, WCC 60WC 23, C 861/6.7, 59/7.061.75/8.25Δ3,5-Δ2,4-Dienoyl-CoA isomeraseQ130112CC 3435.99/6.61DermcidinP816051ENAGEDPGLARM66/7.411.27/6.50Dienoyl-CoA reductaseQ166981VAGHPNIVINNAAGNFISPTERCC 2236.06/9.35Dihydroflavonal 4-reductase, putativeO22617 (F)1LLEHGYTVRWC30/6.238.08/6.70Dihydrolipoamide dehydrogenaseP096227173MM 65M 265/6.954.18/7.59Dihydrolipoamide S-succinyltransferasePN06737223MM 55M 4354/5.748.69/9.01DNA polymerase III, αP57332 (B)1LTLLASTQEGYKNLILLISRMM 34132.78/9.2DNA-directed RNA polymerase IIP249281NSVSQVIQLRWCC 4514.57/6.9EGLN2 proteinQ96KS01LLIFWSDRWC46/6.643.65/8.20Electron transfer flavoprotein, αP138048M, CC 2235.07/8.62Electron transfer flavoprotein, βP381174WC46/6.627.83/8.50Elongation factor, TuP494112CC 3138/6.849.54/7.26Elongation factor, putativeP15252 (T)2CC 8026/5.114.59/5.04EmmprinP356132PP 5029.22/5.41Endoplasmic reticulum proteinP300407302M, WCC 5434/5.228.99/6.77Enolase, αP067338326M, C, WCC 45WC 26, M 47, C 2057/6.6, 53/6.9, 55/6.747.10/6.36Enolase, βP139291AAVPSGASTGIYEALELRCC 2036/5.646.81/8.00Enolase, γP09104195011M, C, WCC 45M 3, C 2749/4.9, 51/4.747.15/4.94Enoyl-CoA hydrataseP300844M, CM 32, C 3231.37/8.34Epoxide hydrolase 1P070994M, CM 45, C 3552.94/6.77Esterase DP107682CC 3531.44/7.0Estradiol 17β-dehydrogenase 4P516591VVLVTGAGAGLGRCC 3579.68/8.96Estradiol 17β-dehydrogenase 8Q925061SALALVTGAGSGIGRCC 3226.97/6.09Fatty acid-binding proteinQ014691FEETTADGRWC28/6.515.16/6.60Fibrinogen, βP026751EGVNDNEEGFFSARCC 7312.73/6.16Fibrinogen, γP026791ANQQFVYCEIDGSGNGWTVFQKWC44/6.551.48/5.40FibronectinP027511TGLDSPTGLDFSDITANSFTVHWIAPWC44/6.8137.42/5.00Flagellar biosynthetic protein, putativeO24978 (H)1IAIGFVGIILIASAIMGRFKCC 3246/6.927.99/6.50Flavin reductaseP300431TVAGQDAVIVLLGTRCC 2940/7.421.98/7.00FMS-related tyrosine kinase 1P53767 (R)1YSVHGYSLIIKMM 4970/5.6150.23/8.7Fodrin αS01091 (X)2PP >9751.94/5.7Fumarate hydrataseP079544CC 44, M 4454.63/8.85GelsolinP063966CC 10098/5.4–5.685.70/5.90Glial fibrillary acidic proteinP1413611283C, WCC 35WC 2960/5.349.88/5.42Glucosamine N-acetyl-6-sulfataseP155861IQEPNTFPAILRCC 2062.08/8.60Glucose transporter 1P111662PP 10053.99/9.16Glucose-regulated protein 75 kDaP3864611210M, P, WCP 74WC 10, M 579/5.3, 78/5.473.78/5.97Glucose-regulated protein 78 kDaP11021244315M, C, P, WCC 75, P 70M 16, P 380/5.0, 83/5.072.33/5.07Glucose-regulated protein 94 kDaP1462514C, PC 10098/5.0, 90/5.192.51/4.78Glutamate dehydrogenase 1P003679247M, C, WCM 32, C 55M 3558/7.161.39/7.66Glutathione S-transferase PP092119492M, C, WCM 23C 2828/5.523.30/5.96Glyceraldehyde-3-phosphate dehydrogenaseP044068488M, C, P, WCM 35, C 35, P 35WC 9, M 4, C 18, P 142/7.4–7.8, 40/7.8–8.5, 40/66–7.035.92/8.58Glycogen phosphorylaseP067371VLYPNDNFFEGKCC 9597.13/7.1GTP-binding protein Rab1TVHUYP2MM 3122.47/5.47GTP-binding protein yptV5P36864 (V)2MM 2923.04/5.8Guanine nucleotide-binding protein, α 2P297771IGAADYQPTEQDILRCC 3839.96/5.59Guanine nucleotide-binding protein, β 1P049014PP 3437.38/5.60Guanine nucleotide-binding protein, β 2P110168234M, WC, PP 35M 3439/5.2–5.437.33/5.6Heat shock cognate 71-kDa proteinP11142132615M, C, WCM 70, C 70WC 6, C 276/5.2, 31/7.070.89/5.37Heat shock protein 105 kDaQ925981GCALQCAILSPAFKCC 10096.15/5.38Heat shock protein 27 kDaP047926335M, P, WCM 34, P 32M 3730/5.922.78/5.95Heat shock protein 60 kDaP1080912311M, WCM 6566/.5.661.05/5.70Heat shock protein 70 B2P41827 (A)3CC 7270.14/5.8Heat shock protein 70-kDa protein 1P081073CC 7269.52/5.00Heat shock protein 90 kDa, αP079005CC 9698/4.884.66/4.94Heat shock protein 90 kDa, βP082385CC 9598/4.883.13/4.97Heat shock-related 70-kDa protein 2P546525CC 7570.00/5.7Hemoglobin, βP020238C, PC 100, P 2015.86/6.81Hemoglobin, δP020423CC 4515.92/7.97Heterogeneous nuclear ribonucleoproteins A2/B1P226261IDTIEIITDRMM 3637.42/8.97Heterogeneous nuclear ribonucleoproteins C1/C2P079101GFAFVQYVNERCC 3633.29/5.11Hexosaminidase AP068651GLETFSQLVWKCC 50, C 5560.69/5.04Hexosaminidase BP076863WC30/5.963.09/6.70High mobility group protein 1P094291IKGEHPGLSIGDVAKWC46/6.624.76/5.70Histone H2A.xP161041VTIAQGGVLPNIQAVLLPKMM 3014.05/10.87Histone H2B.nP235271ESYSIYVYKMM 2013.80/10.32Histone H4P023043PP 1911.24/11.36Hydroxyacyl-CoA dehydrogenaseQ168367CC 23C 3338/7.734.28/8.88Hydroxyisobutyrate dehydrogenaseP319371DFSSVFQFLRMM 3535.32/8.38Hydroxymethylglutaryl-CoA lyaseP359142CC 2334.39/8.81Hydroxymethylglutaryl-CoA synthaseP548688CC 5056.63/8.40Hypothetical protein ypsBP50839 (J)1LSNLEKHVFGSKMM 1441/5.111.57/5.9Hypothetical protein 16.3 kDaO151131VLEEALLSRCC 4697.32/4.9Hypothetical protein 68.9 kDaQ8T845 (D)1IDLDSLPPSLKWCWC 2766/7.168.85/7.0Hypothetical protein MJ0677Q58090 (N)1KFYANRYAELAKMM 4970/5.635.84/5.1Hypothetical protein ykgBO34499 (J)1ALSEPKLAAKCC 1638.39/5.4Ig γ-1 chain C regionP018573MM 4255/7.936.09/8.60Inorganic pyrophosphataseQ151811YVANLFPYKCC 7032.84/5.27Intermediate filament protein ON3P18520 (C)3CC 4057.78/5.15Interphotoreceptor retinoid-binding proteinP1074515239M, C, WCC 100WC 21, M 8, C 35>97/4.7, >97/4.8135.36/4.98Intracellular chloride channel protein p64H1Q96NY71YLNNAYARWC97/4.464.90/4.20Isocitrate dehydrogenase (NAD), αP502132CC 4539.57/6.9Isocitrate dehydrogenase (NADP)P487353CC 4358.74/8.89Isopentenyl diphosphate, Δ-isomerase 1Q139071AFSVFLFNTENKCC 97284.28/5.22Statherin precursorP028081DSSEEKFLRCC 3970/4.65.19/8.70Succinate semialdehyde dehydrogenaseP516491LAGLSAALLRCC 5557.21/8.62Succinyl-CoA ligase, β chainQ96I992CC 4045.13/5.46Succinyl-CoA:3-ketoacid-CoA transferaseP558094CC 6056.15/7.13Superoxide dismutaseP041795341GDVTAQIALQPALKM, CM 25C 1328/6.724.72/8.35T-complex protein 1, αP1798710 (10)246MM 1765/5.7–5.860.34/5.80T-complex protein 1, βP783717 (10)186MM 3357/6.157.49/6.01T-complex Protein 1, εP486436175M, WCM 3662/5.359.67/5.45T-complex Protein 1, γP493689 (20)2MM 3264/6.160.33/6.23T-complex protein 1, θP509907 (18)16MM 2855/5.359.62/5.42T-complex protein 1, ζP402271VATAQDDITGDGTTSNVLIIGELLKMM 5065/6.558.00/6.6Thioredoxin-dependent peroxide reductase 1P300483M, CM 32C 1530/5.8–5.927.69/7.68Thioredoxin-dependent peroxide reductase 2Q0683010416WC, C, PP 31WC 1, C 127/7.4, 28/6.6, 39/7.522.11/8.27Thioredoxin peroxidase 1P321195312M, WC25/5.921.89/5.66Thiosulfate sulfurtransferaseQ167622CC 2233.16/6.78TransaldolaseP378373CC 3637.54/6.36trans-Enoyl-CoA isomeraseP421262CC <1932.81/8.80TransferrinP027879152M, C, WCC 80WC 20, M 786/6.2, 80/6.577.04/6.81Transitional endoplasmic reticulum ATPaseP550724CC 10089.32/5.14TransketolaseP294011LDNLVAILDINRCC 7067.87/7.58TransthyretinP027665 (10)51CC 1237/5.312.60/5.33Trifunctional enzyme, αP409393C, PC 79, P 9083.10/9.28Triosephosphate isomeraseP009389385C, WCC 97117.78/5.57Unknown protein AF007134AF0071345 (17)24CC 835/6.427.97/6.08Unknown protein AAH08028Q96HW26MM 4251/7.849.40/9.4Unknown Protein AAH17450Q8WVW512WC46/6.6, 38/6.340.49/6.1Unknown protein MGC:3932Q96E763MM 5165/7.657.94/8.6Unknown protein MGC:8116Q922U2 (M)3WCWC 560/4.861.75/8.1Vacuolar ATP synthase A, ubiquitous isoformP386064CC 7068.26/5.35Vacuolar ATP synthase, brain isoformP212817172M, CC 50M 2952/5.356.57/5.66Villin 2P1531114PP 7069.40/5.94VimentinP0867014393C, P, WCC 40, P 40WC 360/5.053.65/5.06Vitronectin receptor, αP06756111716M, P, WCP >97M 30>97/4.4116.05/5.45Voltage-dependent anion channel 1P217967386M, P, WCM 35, P 35WC 14, M 6, P 238/7.8, 36/8.2, 40/8.030.64/8.63Voltage-dependent anion channel 2P458804PP 3531.59/7.50a Swiss Protein database accession numbers are shown in plain font. Links to Swiss Protein accession numbers use the EXPASY server at http://us.expasy.org/sprot/. NCBI accession numbers are in italics. Links to NCBI accession numbers use the Entrez server at http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=Protein. Parenthesis following accession numbers designate identifications based on homology with other species: A, Anopheles albimanus; B, Buchuera aphyidicola; C, Carassius auratus; D, Dictyostelium discoideum; F, Fragaria x ananassu; H, Helicobacter pylori; J, Bacillus; L, Amaranthus hypochondriacus; M, Mus musculus; N, Methanocaldococcus jannaschii; Q, Borrelia burgdorferi; R, Rattus norvegicus; T, Hevea brasiliensis; V, Volvox carter; X, Xenopus laevis.b Average error of measured peptide masses is shown for identifications based solely on peptide mass mapping.c Peptide sequences are shown for identifications based on a single peptide (underlined residues were determined).d Cell fractions in which the protein was found: WC, whole cell; C, cytosolic; P, P2 membrane; M, microsomal.e SDS-PAGE are shown for C, P2, and M cell fractions in electronic Fig. 5. Identified proteins were excised from the indicated molecular mass region.f Representative 2D PAGE profiles for WC, C, P2, and M cell fractions are shown in electronic Fig. 1, Fig. 2, Fig. 3, Fig. 4, respectively. Identified proteins were excised from the numbered spots in the figures and/or from the indicated molecular mass and pI range in other 2D gels. Open table in a new tab Fig. 2Human RPE cytosol. Approximately 150 μg of RPE cytosolic protein were applied to 2D gel analysis (87,550 V-h IEF), and the gel was stained with silver. Numbered spots were excised for protein identification (Table I).View Large Image Figure ViewerDownload (PPT)Fig. 3Human RPE P2 membrane subcellular fraction. Approximately 80 μg of RPE P2 membrane protein were applied to 2D gel analysis (87,550 V-h IEF), and the gel was stained with silver. Numbered spots were excised for protein identification (Table I).View Large Image Figure ViewerDownload (PPT)Fig. 4Human RPE microsome subcellular fraction. Approximately 190 μg of RPE microsomal protein were applied to 2D gel analysis (88,400 V-h IEF), and the gel was stained with colloidal Coomassie Blue. Numbered spots were excised for protein identification (Table I).View Large Image Figure ViewerDownload (PPT)Fig. 5SDS-PAGE of human RPE subcellular fractions. One-dimensional SDS-PAGE was performed with the RPE P2 membrane (∼30 μg) and the microsomal fractions (∼30 μg) on a minigel (10 × 8 cm) and with the RPE cytosolic fraction (∼40 μg) on a 20- × 20-cm gel. Gels were stained with colloidal Coomassie Blue, and consecutive ∼1-mm bands were excised from the top to the bottom of the lane for protein identification (Table I). A total of 65 bands was cut and analyzed from each of the P2 and microsomal sample lanes, and 80 bands were cut from the cytosolic sample lane.View Large Image Figure ViewerDownload (PPT) In addition to housekeeping proteins common to many cell types, a number of proteins were identified that are known to be associated with specialized functions of the RPE. These included proteins associated with retinoid metabolism and the visual cycle proteins such as cellular retinaldehyde-binding protein, cellular retinol-binding protein, retinal pigment epithelium 65, 11-cis-retinol dehydrogenase 5, retinal G protein-coupled receptor, and interphotoreceptor retinoid-binding protein. Interphotoreceptor retinoid-binding protein is synthesized in the photoreceptor cells; however, it appears to be part of a visual cycle protein complex in the RPE (13.Bhattacharya S.K. Wu Z. Jin Z. Yan L. Miyagi M. West K. Nawrot M. Saari J.C. Crabb J.W. Proteomic approach to identification of a mammalian visual cycle protein complex.FASEB J. 2002; 16 (Abstr. 16.2): A14Google Scholar). The RPE is the most active phagocytic tissue in humans, and each RPE cell daily phagocytizes the shed outer segments tips from ∼50 photoreceptor cells. Identified proteins involved in macromolecular degradation included cathepsins B, D, and Z; lysozyme; and several proteasome components. The photooxidative environment in the retina and active phagocytic processing provide abundant reactive oxygen species to the RPE. Identified antioxidant proteins included thioredoxin-dependent peroxide reductase 1 and 2, catalase, peroxiredoxin 6, superoxide dismutase, glutathione S-transferase, and thioredoxin peroxidase. Ten of the 278 identified proteins are currently designated hypothetical or of unknown function, and ∼6% were identified based on homology with other species. Despite significant efforts to purify RPE cells free of extracellular debris and other cell types, low level blood contamination (e.g. hemoglobin) was detected in three gels. Photoreceptor-specific proteins such as phosducin, recoverin, and rhodopsin were also identified and may be in the RPE due to phagocytosis. The RPE proteins identified here provide an initial reference library for targeted studies of this important visual tissue.
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