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Evidence That Light Modulates Protein Nitration in Rat Retina

2002; Elsevier BV; Volume: 1; Issue: 4 Linguagem: Inglês

10.1074/mcp.m100034-mcp200

1535-9484

Masaru Miyagi, Hirokazu Sakaguchi, Ruth M. Darrow, Yan Lin, Karen A. West, Kulwant S. Aulak, Dennis J. Stuehr, Joe G. Hollyfield, Daniel T. Organisciak, John W. Crabb,

Retinal Diseases and Treatments

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4–33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology. As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4–33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology. Nitration of tyrosine is one of several protein modifications that can occur as a result of oxidative stress (1.Haddad I.Y. Pataki G. Hu P. Galliani C. Beckman J.S. Matalon S. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.J. Clin. Invest. 1994; 94: 2407-2413Google Scholar). A number of inflammatory and neurodegenerative disorders have been associated with tyrosine nitration including ocular inflammation, retinal ischemia, lung infection, cancer, Parkinson's disease, Alzheimer's disease, and Huntington's disease (2.Halliwell B. Zhao K. Whiteman M. Nitric oxide and peroxynitrite. The ugly, the uglier and the not so good: a personal view of recent controversies.Free Radic. Res. 1999; 31: 651-669Google Scholar). The abundancy of this protein modification is low, perhaps less than 1 in 106 tyrosine (3.Shigenaga M.K. Lee H.H. Blount B.C. Christen S. Shigeno E.T. Yip H. Ames B.N. Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3211-3216Google Scholar), and identification of all in vivo protein nitration targets have utilized antibodies directed against nitrotyrosine (4.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). The only successful identification of in vivo sites of tyrosine nitration to date has been achieved with histone H2B from mouse tumors, a relatively abundant protein that may accumulate more nitrotyrosine than the average cellular protein because of greater stability and lower turnover (5.Haqqani A.S. Kelly J.F. Birnboim H.C. Selective nitration of histone tyrosine residues in vivo in mutatect tumors.J. Biol. Chem. 2002; 277: 3614-3621Google Scholar). Previously we used a combination of immunological detection and MALDI-TOF 1The abbreviations used are: MALDI-TOF, matrix assisted laser desorption ionization time of flight; BSA, bovine serum albumin; IEF, isoelectric focusing; LC, liquid chromatography; MS/MS, tandem mass spectrometry; NO, nitric oxide; nY, nitrotyrosine; PBS, phosphate-buffered saline; PVDF: polyvinylidene difluoride; RPE, retinal pigment epithelium; ROS, rod outer segments; 2D, two-dimensional; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. MS to identify proteins nitrated on tyrosine during inflammatory challenge (4.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) and now focus on a possible link between protein nitration and retinal light damage. Partial protection from retinal light damage has been observed in rats treated with N-nitroarginine methyl ester (6.Goureau O. Jeanny J.C. Becquet F. Hartmann M.P. Courtois Y. Protection against light-induced retinal degeneration by an inhibitor of NO synthase.Neuroreport. 1993; 5: 233-236Google Scholar), an inhibitor of nitric-oxide synthase. Because nitric-oxide synthase provides the in vivo precursor for nitrating agents, nitration may play a role in light-induced retinal degeneration. Retinal damage from intense visible light was reported first in 1966 (7.Noell W.K. Walker V.S. Kang B.S. Berman S. Retinal damage by light in rats.Invest. Ophthalmol. Vis. Sci. 1966; 5: 450-473Google Scholar) yet the molecular mechanisms remain poorly understood. Light-induced photoreceptor cell degeneration appears to involve both apoptosis and necrosis (8.Organisciak D.T. Winkler B.S. Osborne N.N. Chader G.J. Progress in Retinal and Eye Research. 13. Pergamon Press, Oxford1994: 1-29Google Scholar, 9.Hafezi F. Steinbach J.P. Marti A. Munz K. Wang Z.Q. Wagner E.F. Aguzzi A. Reme C.E. The absence of c-fos prevents light-induced apoptotic cell death of photoreceptors in retinal degeneration in vivo.Nat. Med. 1997; 3: 346-349Google Scholar, 10.Organisciak D.T. Darrow R.A. Barsalou L. Darrow R.M. Lininger L.A. Light-induced damage in the retina differential effects of dimethylthiourea on photoreceptor survival, apoptosis and DNA oxidation.Photochem. Photobiol. 1999; 70: 261-268Google Scholar) via a process initiated by oxidative stress, because antioxidants such as ascorbate and dimethylthiourea provide protection (8.Organisciak D.T. Winkler B.S. Osborne N.N. Chader G.J. Progress in Retinal and Eye Research. 13. Pergamon Press, Oxford1994: 1-29Google Scholar, 10.Organisciak D.T. Darrow R.A. Barsalou L. Darrow R.M. Lininger L.A. Light-induced damage in the retina differential effects of dimethylthiourea on photoreceptor survival, apoptosis and DNA oxidation.Photochem. Photobiol. 1999; 70: 261-268Google Scholar). Damaging light is thought to produce reactive oxygen species; however the identity of such species and the pathways of oxidative damage remain unknown (10.Organisciak D.T. Darrow R.A. Barsalou L. Darrow R.M. Lininger L.A. Light-induced damage in the retina differential effects of dimethylthiourea on photoreceptor survival, apoptosis and DNA oxidation.Photochem. Photobiol. 1999; 70: 261-268Google Scholar). The similarity in the action spectrum of retinal light damage and the absorption spectrum of rhodopsin (∼500 nm maximum) has lead to the hypothesis that injury may be initiated by rhodopsin bleaching (7.Noell W.K. Walker V.S. Kang B.S. Berman S. Retinal damage by light in rats.Invest. Ophthalmol. Vis. Sci. 1966; 5: 450-473Google Scholar, 11.Williams T.P. Howell W.L. Action spectrum of retinal light-damage in albino rats.Invest. Ophthalmol. Vis. Sci. 1983; 24: 285-287Google Scholar). Additional evidence supports the involvement of retinoids in the process. Blue light induces apoptosis in cultured retinal pigment epithelial cells in a manner related directly to the content of pyridinium bisretinoid, i.e. lipofuscin component A2E (12.Sparrow J.R. Nakanishi K. Parish C.A. The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.Invest. Ophthalmol. Vis. Sci. 2000; 41: 1981-1989Google Scholar). In vitro, light induces aggregation of the photoreceptor-specific protein ABCR in the presence of all-trans-retinal (13.Sun H. Nathans J. ABCR, the ATP-binding cassette transporter responsible for Stargardt macular dystrophy, is an efficient target of all-trans-retinal-mediated photooxidative damage in vitro. Implications for retinal disease.J. Biol. Chem. 2001; 276: 11766-11774Google Scholar). Dietary vitamin A-depleted rats (14.Noell W.K. Albrecht R. Irreversible effects on visible light on the retina: role of vitamin A.Science. 1971; 172: 76-79Google Scholar), as well as animals with impaired visual cycle retinoid processing proteins such as CRALBP (15.Saari J. Nawrot M. Kennedy B.N. Garwin G.G. Hurley J.B. Huang J. Possin D.E. Crabb J.W. Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation.Neuron. 2001; 29: 739-748Google Scholar), RPE65 (16.Wenzel A. Reme C.E. Williams T.P. Hafezi F. Grimm C. The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration.J. Neurosci. 2001; 21: 53-58Google Scholar), and RDH5 (17.Sieving P.A. Chaudhry P. Kondo M. Provenzano M. Wu D. Carlson T.J. Bush R.A. Thompson D.A. Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1835-1840Google Scholar), appear to be more resistant to light damage than normal control animals. However age, diet, genetics, and environmental light history all influence the extent and type of retinal injury from light exposure (8.Organisciak D.T. Winkler B.S. Osborne N.N. Chader G.J. Progress in Retinal and Eye Research. 13. Pergamon Press, Oxford1994: 1-29Google Scholar). The purpose of this study was to improve methods for characterizing protein nitration and to investigate whether protein nitration is a mediator in photoreceptor light damage. An improved 2D gel Western method for detecting nitrated proteins is described, and a quantitative baseline is established for LC MS/MS determination of sites of tyrosine nitration. We have also surveyed the nitroproteome of retina from rats exposed to intense light or maintained in the dark. The results indicate that the distribution and identity of nitrotyrosine-containing proteins within the cell layers of the retina changes with light exposure. Albino male Sprague-Dawley rats (Harlan Inc., Indianapolis, IN) were received at 21 days of age and maintained in darkness until use. The dark environment was interrupted with dim red illumination about 30 min per day during routine animal care. The rats were fed a standard rat chow (Teklad, Madison, WI) and given water ad libitum. Rats maintained in a dark environment for 2–4 months were exposed to intense (1500 lux) green light (490–580 nm) for 3 h and sacrificed immediately following light treatment under dim red illumination in a chamber with a CO2-saturated atmosphere. All procedures involving rats followed the protocols outlined in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Four animals were used for 2D gel Western analyses of retinal proteins, and eight animals were used for immunocytochemistry. Retinas were excised from rats within 2 min of death as described by Delmelle et al. (18.Delmelle M. Noell W.K. Organisciak D.T. Hereditary retinal dystrophy in the rat: rhodopsin, retinol, vitamin A deficiency.Exp. Eye Res. 1975; 21: 369-380Google Scholar), rinsed in PBS, and frozen in liquid nitrogen. Photoreceptor rod outer segments (ROS) were isolated from retinas using a discontinuous sucrose gradient technique as described by Organisciak et al. (19.Organisciak D.T. Wang H. Kou A.L. Rod outer segment lipid-opsin ratios in the developing normal and retinal dystrophic rat.Exp. Eye Res. 1982; 34: 401-412Google Scholar) and stored at −80°C until use. Each frozen retina was homogenized in 50 μl of isoelectric focusing (IEF) solvent B (7 m urea, 2 m thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholite, pH 3–10, 1% dithiothreitol) using a disposable pestle at 4°C. The homogenate was centrifuged at 14,000 rpm/min (Eppendorf table tap centrifuge 5415C) for 30 min, and the supernatant was collected. Photoreceptor ROS were incubated in PBS containing 1% dodecyl-β-maltoside at 4°C for 1 h. Sucrose in the ROS solution was removed by repeated concentration and dilution with the same buffer using an ultracentrifuge filter tube (5-kDa molecular mass cut-off; Millipore, Bedford, MA). Lipids were extracted from ROS with an equal volume of chloroform:methanol (2:1 v/v), and proteins in the aqueous phase were collected, dried, and redissolved in IEF solvent B. Proteins in retinal and ROS extracts were quantified by a modified Bradford procedure (20.Ramagli L.S. Quantifying protein in 2-D PAGE solubilization buffers.Methods Mol. Biol. 1999; 112: 99-103Google Scholar). For one-dimensional gel electrophoresis, retinal extracts and ROS preparations in solvent B were diluted 1:1 with Laemmli SDS-PAGE sample buffer, and electrophoresis was performed according to Laemmli (21.Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Google Scholar) using the Bio-Rad Mini-Protein II system. Two-dimensional gel electrophoresis was performed with the IPGphor/IsoDalt systems (Amersham Biosciences) as described by West et al. (22.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). First dimension IEF was performed with the IPGphor system in IEF solvent B using 18 cm of non-linear pH 3–10 immobilized pH gradient strips and a programmed voltage gradient (22.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). After the first dimension IEF, the strips were incubated in 10 ml of reducing solution (50 mm Tris-HCl, pH 8.8, 6 m urea, 100 mm dithiothreitol, 30% glycerol, 2% SDS) for 15 min at room temperature and then in 10 ml of alkylation solution (50 mm Tris-HCl, pH 8.8, 6 m urea, 250 mm iodoacetoamide, 30% glycerol, 2% SDS) for 15 min at room temperature. For the second dimension, the immobilized pH gradient strips were embedded in 0.7% (w/v) agarose on the top of 12% acrylamide slab gels (23.5 × 18.0 × 0.15 cm) containing a 4% stacking gel, and SDS-PAGE was performed overnight at 20–30 mA/gel with the IsoDalt system (22.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). Gels were stained with colloidal Coomassie Blue (Pierce Code Blue) according to the vendor. Detection limits of the stain were determined to be ∼15 ng of protein on 1.5-mm-thick gels based on quantitative analyses with BSA (quantified by amino acid analysis) and a commercial preparation of molecular weight markers (catalogue number 161–0304; Bio-Rad). For Western blot analysis, proteins in 2D gels were partially transferred to PVDF membranes (Millipore, Bedford, MA) at 320 mA/gel for 25 min using a Semi Dry Transfer Cell (Bio-Rad). Proteins remaining in the gels were visualized by colloidal Coomassie Blue and excised for identification. Two PVDF membranes were used for each set of Western analysis. One membrane was chemically reduced before Western analysis, and another membrane was used for Western analysis without reduction. Reduction of nitrotyrosine to aminotyrosine was achieved by treating one membrane with 10 mm sodium dithionite in 50 mm pyridine-acetate buffer, pH 5.0, for 1 h at room temperature. After the reaction, the membrane was rinsed with distilled water and then equilibrated with wash solution 1 (20 mm Tris, 150 mm NaCl, pH 7.5, 0.2% Tween 20). The reduced and non-reduced PVDF membranes were blocked for 1 h at room temperature in blocking solution (20 mm Tris, 150 mm NaCl, pH 7.5, 0.2% Tween 20, 1% bovine serum albumin). The membranes were then probed as before (4.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) with a monoclonal antibody against 3-nitrotyrosine (1:3,000 dilution; Upstate Biotechnology, Lake Placid, NY) for 2 h at 4°C in blocking solution. The membranes were then washed five times in wash solution 1 and probed with horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Amersham Biosciences) for 1 h at room temperature in blocking solution. After washing the membranes five times with wash solution 2 (20 mm Tris, 150 mm NaCl, pH 7.5), immunoreactive proteins were visualized on x-ray film using a chemiluminescent protein detection system (Amersham Biosciences). Nitrated BSA was used as a positive standard protein. X-ray film and gels were scanned with a GS-710 imaging densitometer (Bio-Rad), and the intensity of chemiluminescence and of Coomassie Blue staining was measured using PDQest 2D Gel Analysis Software, Version 6 (Bio-Rad). BSA was nitrated by incubating the protein (10 μm) with 50 mm tetranitromethane in 100 μl of 50 mm Tris-HCl, pH 8.0, at room temperature. After 5 min, the reaction was stopped by applying the preparation to a gel filtration column (Sephadex G-25, 1 × 5 cm; Pharmacia Biotech) equilibrated in 0.1% formic acid. The nitrated and desalted BSA was quantified by phenylthiocarbamyl amino acid analysis using an Applied Biosystems model 420H/130/920 automated analysis system (23.Crabb J.W. West K.A. Dodson W.S. Hulmes J.D. Coligan J.E. Ploegh H.L. Smith J.A. Speicher D.W. Current Protocols In Protein Science. John Wiley & Sons, Inc., New York1997: 11.9.1-11.9.42Google Scholar). Nitrated BSA (30 μg) was added to 200 mm ammonium bicarbonate, pH 8, containing 8 m urea, diluted 1:4, and digested with 1 μg of trypsin (Promega, Madison, WI) at 37°C overnight under argon. Identification of anti-nitrotyrosine reactive proteins by peptide mass mapping was as described previously (4.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, 22.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). To facilitate localization of immunoreactive spots in stained gels, Western profiles were transferred to a transparency with an office copy machine and enlarged to the size of the gel after staining. By overlaying the Western transparency on the stained gel, immunoreactivity on the membrane was correlated with specific gel spots. To determine how much to enlarge the Western blot transparency, gel dimensions were measured on the PVDF membrane after the blotting and compared with gel dimensions after staining. Briefly, the 2D gel spots were excised, Coomassie Blue was washed away, and proteins were digested in-gel with trypsin (Promega, Madison, WI) in 15 mm N-ethylmorpholine acetate, pH 8.6. Peptides were extracted from in-gel digests with 60% acetonitrile containing 0.1% trifluoroacetic acid, dried in a Speed Vac, redissolved in 0.1% trifluoroacetic acid, and adsorbed onto C18 ZipTips (Millipore, Bedford, MA). Following ZipTip elution with 7 μl of 75% acetonitrile, 0.02% trifluoroacetic acid, peptides were subjected (1 μl) to mass spectrometric analysis 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 NCBI sequence databases for matches using MS-Fit and Profound search programs. All searches were performed with a mass tolerance of 50 ppm. Positive identification required a minimum of five peptide matches and the highest probability ranking in both of the search programs employed. LC MS/MS was also used to analyze immunoreactive gel spots and nitrated BSA. Tryptic digests of gel spots (≤6 μl) were diluted with 0.1% formic acid (10 μl), and 10 μl were injected by autosampler onto a 0.3 × 1-mm trapping column (PepMap C18; LC Packings) using a CapLC system (Micromass), a switching valve (Micromass), and a flow rate of 5 μl/min. Solvent pumps were set at 8 μl/min, and the flow rate was controlled with a splitter in front of the switching valve. Peptides were eluted at 250 nl/min and chromatographed on a 50-μm × 5-cm Biobasic C18 column (New Objective, Cambridge, MA) with a gradient of 5–40% acetonitrile over 20 min followed by 80% acetonitrile for 5 min. The eluent was direct into a quadrupole time-of-flight mass spectrometer (QTOF2; Micromass, Beverly, MA) and ionized immediately using an electrosprayer designed in-house. The mass spectrometer was operated in standard MS/MS switching mode with the three most intense ions in each survey scan subjected to MS/MS analysis. In addition, the "include function" of the instrument operating software was used to program MS/MS analysis of precursor ions of all possible nitrotyrosine-containing tryptic peptides based on the structure of the proteins preidentified by MALDI-TOF MS (calculated as doubly and triply charged ions). Protein identifications and MS/MS data analyses utilized Micromass software ProteinLynx™ Global Server, MassLynx™ Version 3.5, and the Swiss-Protein and NCBI protein sequence databases (January 2002). MS/MS spectra of possible nitrated peptides were examined manually to determine sites of modification. The relative amount of nitration at each nitration site in BSA was estimated based on the ion intensity of the nitrotyrosine-containing peptides, essentially as described for estimating relative amounts of glycosylation (24.Kapron J.T. Hilliard G. Lakins J. Tenniswood M. West K.A. Carr S.A. Crabb J.W. Identification and characterization of glycosylation sites in human serum clusterin.Protein Sci. 1997; 6: 2120-2133Google Scholar). First the observed ion intensities for each charge state of a nitrated peptide were summed and then the total intensity for each nitrated plus unmodified peptide was calculated. The relative amount of nitration at the site was estimated as percent of the total intensity. Eyes enucleated immediately after the light exposure were used for immunocytochemical analyses (25.Hollyfield J.G. Rayborn M.E. Nishiyama K. Shadrach K.G. Miyagi M. Crabb J.W. Rodriguez I.R. Interphotoreceptor matrix in the fovea and peripheral retina of the primate Macaca mulatta: distribution and glycoforms of SPACR and SPACRCAN.Exp. Eye Res. 2001; 72: 49-61Google Scholar). After separating the anterior segment, posterior eye cups were fixed in 4% paraformaldehyde in PBS for 4 h at 4°C. The eyes were then cryoprotected in 30% sucrose overnight and embedded in optimal cutting temperature compound. Cryosections (16-μm) were prepared on gelatin-coated slides and stored at −80°C until use. Rats without light exposure were used as control animals. After air drying, sections were washed in PBS for 10 min and then incubated in PBS containing 5% BSA (Sigma) and 0.3% Triton X-100 for 1.5 h. The sections were then probed with mouse monoclonal anti-nitrotyrosine antibody (1:100 dilution; Upstate Biotechnology, Lake Placid, NY) in PBS containing 0.2% BSA and 0.2% Tween 20 overnight at 4°C. Fluorescein-conjugated anti-mouse IgG was used as secondary antibody (1:200 dilution, 1 h incubation at 4°C; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Sections were mounted with mounting medium (Vector Laboratories Inc., Burlingame, CA), and normal mouse IgG was used as a control for the primary antibody. A total of 16 eyes (8 light-exposed and 8 control) were analyzed with a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems Inc., Heidelberg, Germany). Images were captured from the posterior area of the retina, and more than 20 sections were examined per eye. The intensity of immunohistochemical reactivity was quantified using Quantity One software (Bio-Rad) after converting the color images from the confocal microscope to grayscale images in Adobe PhotoShop. To distinguish between specific and nonspecific nitrotyrosine immunoreactivity, we modified previously described methodology (4.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) and performed Western analyses before and after converting protein nitrotyrosine to aminotyrosine, as summarized in Fig. 1. Chemical reduction was achieved by treating PVDF-immobilized proteins with 10 mm sodium dithionite in 50 mm pyridine acetate, pH 5.0, at room temperature for 1 h. Before reduction, nitrated BSA exhibited strong nitrotyrosine immunoreactivity in Western analysis, and after reduction, the protein exhibited no immunoreactivity (Fig. 2, lanes 2 and 4). Western analyses of rat retinal proteins with and without dithionite reduction are also shown in Fig. 2 (lanes 1 and 3). Two bands, ∼55 and 60 kDa, retain immunoreactivity after dithionite treatment, suggesting that these are false positives. Thus, Western analysis before and after dithionite reduction of membrane-immobilized proteins facilitates the identification of specific and nonspecific anti-nitrotyrosine antibody recognition.Fig. 2Western analysis before and after reduction of nitrotyrosine to aminotyrosine. A retinal extract from a light-exposed 2-month-old rat (lanes 1 and 3, 20 μg/lane) and nitrated BSA standard (lanes 2 and 4, 20 ng/lane) were electrophoresed on 10% SDS-polyacrylamide gels, blotted to PVDF membrane, and probed with a monoclonal anti-nitrotyrosine antibody. Western analysis results are shown without (lanes 1 and 2) and after (lanes 3 and 4) membrane treatment with dithionite.View Large Image Figure ViewerDownload (PPT) Albumin was modified chemically with tetranitromethane, and sites of nitration were identified and quantified by LC MS/MS analysis of tryptic peptides to evaluate the detection capabilities of our experimental approach. Analyses of the nitrated BSA tryptic digest yielded identification of four sites of tyrosine nitration, namely Tyr161, Tyr173, Tyr357, and Tyr424 (Fig. 3). Unmodified peptides containing each of these residues were also detected. The relative amount of nitration at each site was estimated from four independent LC MS/MS analyses of 1–2 pmol of BSA tryptic digest to be ∼84% at Tyr161, ∼44% at Tyr173, ∼5% at Tyr357, and ∼75% at Tyr424. Detection sensitivity of nitrotyrosine-containing peptides was evaluated by LC MS/MS analysis of 1–1000 fmol of the nitrated BSA tryptic digest. The limits of detection for nitrotyrosine-containing peptides from this preparation of BSA were determined to be 4 to 33 fmol (see Table I, and see Fig. 3).Table IDetection of tyrosine nitration sites in tetranitromethane-modified BSAAmount analyzed (BSA tryptic digest)Total peptides identifiedAmount of nitrotyrosine peptide detectednY161nY173nY357nY424ngfmolfmol6610002284044050750172502221011013188132002016888150575186333563501542380.7108880.35744<0.111 Open table in a new tab Retinas were isolated from a rat exposed to intense green light and another rat maintained in the dark, retinal proteins were fractionated by 2D gel electrophoresis, and nitr