Proteomic Identification of Oxidatively Modified Retinal Proteins in a Chronic Pressure-Induced Rat Model of Glaucoma

Proteomic Identification of Oxidatively Modified RetinalProteins in a Chronic Pressure-Induced Rat Modelof Glaucoma

Gulgun Tezel,1,2 Xiangjun Yang,1 and Jian Cai3

PURPOSE. Based on the evidence of an amplified production ofreactive oxygen species (ROS) during glaucomatous neurode-generation, proteomic analysis was performed to determineoxidative modification of retinal proteins after experimentalelevation of intraocular pressure (IOP).

METHODS. IOP elevation was induced in rats by hypertonicsaline injections into episcleral veins. Protein expression wasdetermined by two-dimensional polyacrylamide gel electro-phoresis (2D-PAGE) of retinal protein lysates obtained fromeyes matched for the cumulative IOP exposure and axon loss.To determine protein oxidation levels, protein carbonyls weredetected through 2D-oxyblot analysis of 2,4-dinitrophenylhy-drazine (DNPH)-treated samples using an anti-DNP antibody.For identification of oxidized proteins, peptide masses wereanalyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chroma-tography-tandem mass spectrometry (LC/MS/MS). In additionto use of different engines in a bioinformatic database searchand performance of peptide sequencing and 2D-Western blotanalysis for confirmation of the identified proteins, immuno-histochemistry was used for further validation of the proteomicfindings.

RESULTS. Comparison of 2D-oxyblots with Coomassie Blue–stained 2D-gels revealed that approximately 60 protein spotsobtained with retinal protein lysates from ocular hypertensiveeyes (of �400 spots) exhibited protein carbonyl immunoreac-tivity, which reflects oxidatively modified proteins. There wasa significant increase in anti-carbonyl reactivity in individualprotein spots obtained with retinal protein lysates from ocularhypertensive eyes compared with the control (P � 0.01). Theidentified proteins through peptide mass fingerprinting andpeptide sequencing included glyceraldehyde-3-phosphate de-hydrogenase, a glycolytic enzyme; HSP72, a stress protein; andglutamine synthetase, an excitotoxicity-related protein. Immu-nolabeling of retina sections with specific antibodies demon-strated cellular localization of these proteins as well as retinal

distribution of the increased protein carbonyl immunoreactiv-ity in ocular hypertensive eyes.

CONCLUSIONS. The findings of this in vivo study provide novelevidence for oxidative modification of many retinal proteins inocular hypertensive eyes and identify three specific targets ofretinal protein oxidation in these eyes, thereby supporting theassociation of oxidative damage with neurodegeneration inglaucoma. By using a proteomic approach, this study alsoexemplifies that proteomics provide a very promising way toelucidate pathogenic mechanisms in glaucoma at the proteinlevel. (Invest Ophthalmol Vis Sci. 2005;46:3177–3187) DOI:10.1167/iovs.05-0208

Mitochondria have been found to be essential in control-ling cell death.1 Involvement of mitochondria during ap-

optosis starts a chain of events that lead to the activation of aproteolytic caspase cascade. In addition, mitochondrial dys-function results in the compromise of mitochondrial oxidativephosphorylation and production of reactive oxygen species(ROS).2,3 Mitochondrial dysfunction has been associated withneuronal apoptosis in an experimental rat glaucoma model,4,5

and free radical scavenging when combined with trophic fac-tors has provided neuroprotection in ocular hypertensive rateyes.6 In agreement with these findings, retinal ganglion cells(RGCs) have been shown to be susceptible to ROS,7 and thesurvival of axotomized neonatal RGCs has been found to becritically sensitive to the oxidative redox state.8 Our recent invitro studies of primary cultures of RGCs have provided furtherevidence that the RGC death induced by different glaucoma-tous stimuli involves both caspase-dependent and caspase-in-dependent components of the mitochondrial cell death path-way, including the generation of ROS. In addition, these invitro studies revealed that antioxidant treatment improves thesurvival of caspase-inhibited RGCs by increasing the intrinsicability of these neurons to survive cytotoxic consequences ofmitochondrial dysfunction.9

In addition to alterations identified in the expression levelof various proteins in glaucomatous eyes,10–14 it is also evidentthat many retinal proteins undergo proteolytic cleavage duringglaucomatous neurodegeneration15–17 or exhibit posttransla-tional modifications, including phosphorylation.13,18 Novelfunctions of proteins after such modifications have importantimplications in the pathogenic mechanisms of glaucoma. Ourrecent in vitro studies have also demonstrated the inconsis-tency between gene and protein expression in RGCs and glialcells, mainly due to protein phosphorylation after exposure toglaucomatous stimuli.18 Because the ultimate function of thegene that resides in the protein can be modulated by posttrans-lational modifications,19,20 studying global changes in the pro-teome (PROTEins expressed by the genOME) can providemore relevant pathophysiological information.

Based on the evidence demonstrating an amplified produc-tion of ROS during glaucomatous neurodegeneration,9 we hy-pothesized that the “oxidative modification” may be anotherposttranslational modification of retinal proteins involved inthe neurodegenerative process of glaucoma. Given that the

From the Departments of 1Ophthalmology and Visual Sciences,2Anatomical Sciences and Neurobiology, and 3Pharmacology and Tox-icology, University of Louisville School of Medicine, Louisville, Ken-tucky.

Supported in part by National Eye Institute Grant R01EY013813(GT) and an unrestricted grant to the University of Louisville Depart-ment of Ophthalmology and Visual Sciences from Research to PreventBlindness. GT is a recipient of the Research to Prevent Blindness SybilB. Harrington Special Scholar Award.

Submitted for publication February 16, 2005; revised April 15 andMay 9, 2005; accepted July 11, 2005.

Disclosure: G. Tezel, None; X. Yang, None; J. Cai, NoneThe publication costs of this article were defrayed in part by page

charge payment. This article must therefore be marked “advertise-ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Gulgun Tezel, University of LouisvilleSchool of Medicine, Kentucky Lions Eye Center, 301 E. Muhammad AliBoulevard, Louisville, KY 40202; [email protected].

Investigative Ophthalmology & Visual Science, September 2005, Vol. 46, No. 9Copyright © Association for Research in Vision and Ophthalmology 3177

identification of alterations in protein complement throughproteomic approaches can help to understand impaired cellu-lar mechanisms, we performed proteomic analysis to deter-mine whether retinal proteins are oxidatively modified duringglaucomatous neurodegeneration in ocular hypertensive eyesand, if so, what the targets are for protein oxidation in theseeyes. Immunochemical detection of protein carbonyls using2D-oxyblot analysis coupled with peptide mass fingerprintingand peptide sequencing revealed oxidative modification ofmany retinal proteins in ocular hypertensive eyes and identi-fied three specific targets of protein oxidation in the retinas ofthese eyes. These novel findings support the association ofoxidative damage with neurodegeneration in glaucoma in vivoand exemplify that proteomics provide a very promising wayto elucidate pathogenic mechanisms of glaucomatous neuro-degeneration at the protein level.

MATERIALS AND METHODS

Experimental Rat Glaucoma Model

Intraocular pressure (IOP) elevation was unilaterally induced in maleBrown Norway rats with an average weight of 200g by hypertonicsaline injections into episcleral veins, as previously described.21 All theanimals were handled according to the regulations of the InstitutionalAnimal Care and Use Committee, and all procedures adhered to thetenets of the ARVO Statement for the Use of Animals in Ophthalmicand Vision Research. Briefly, in rats under general and topical anesthe-sia, with placement of a plastic ring around the equator, a glassmicropipette (attached to a polyethylene tube connected to a tuber-culin syringe) was inserted into a circumferential limbal vein, andapproximately 0.1 mL of 1.75 M saline was injected into the venoussystem. The injection was repeated 1 week later. Unilateral elevation ofIOP was induced, and the fellow eyes with normal IOP served as thecontrol. Intraocular pressure was measured in both eyes immediatelybefore and after saline injection, and the measurements were repeatedin awake animals22 twice per week using a calibrated hand-held tonom-eter (Tonopen; Medtronic Solan, Jacksonville, FL). Animals were ex-cluded from the experiments, if there was no measurable IOP expo-sure, as previously described.23

During a follow-up period of up to 12 weeks after saline injection,IOPs were higher in experimental than in control eyes. Figure 1Ademonstrates the course of IOP elevation over time. The percentageaxon loss presented in Figure 1B was expressed by comparing theaxon count in the ocular hypertensive eye relative to the control felloweye, as will be described later. The degree of cumulative IOP exposurewas estimated by first integrating IOP over time in the ocular hyper-tensive eye, then subtracting the IOP-time integral from that in thenormotensive fellow eye (expressed in units of mm Hg-days), as pre-viously described.23 As shown in Figure 1C, a dose–response effect ofpressure on axon loss was detectable with a threshold for axon loss ata value of approximately 200 mm Hg-days. These findings are consis-tent with previous studies in which the same model was used.21,23,24

Retinas were dissected from the enucleated eyes of animals killed atdifferent time points, and the proteomic experiments described hereinwere performed with retinal protein lysates obtained from ocularhypertensive and control eyes matched for the cumulative IOP expo-sure and axon loss. Specifically, the retinal protein samples obtainedfrom moderately damaged eyes with a cumulative IOP exposure of 200to 400 mm Hg-days were used in this study. As shown in Figure 1, thislevel of cumulative IOP exposure corresponds to a relative axon loss ofno more than 50%.

Optic Nerve Axon Counts

Optic nerve axons were counted with a protocol similar to thatpreviously used.9 For axon counts, optic nerves excised from enucle-ated eyes were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer(pH 7.4) overnight and postfixed in 2% osmium tetroxide for 1 hour.

FIGURE 1. Experimental rat model of chronic pressure-induced glau-coma. (A, B) The course of IOP elevation and axon loss, respectively,during an experimental period of up to 12 weeks after hypertonicsaline injection into the limbal veins in rats. Data are presented as themean � SD. The percentage axon loss was determined by comparingthe estimated total number of optic nerve axons in the ocular hyper-tensive eye relative to the control fellow eye. The degree of cumulativeIOP exposure was estimated by first integrating IOP over time in theocular hypertensive eye, then subtracting the IOP-time integral fromthat in the normotensive fellow eye (mm Hg-days). (C) The associationof axon loss with cumulative IOP exposure.

3178 Tezel et al. IOVS, September 2005, Vol. 46, No. 9

Dehydrated tissues were embedded in epoxy resin, and 1-�m crosssections of the myelinated optic nerves were stained with 1% ToluidineBlue. Captured images were analyzed on computer (Axiovision soft-ware; Carl Zeiss Meditec, Inc., Thornwood, NY), as previously de-scribed.21,25 Briefly, the axon density was counted in randomizedregions of the optic nerve by using a systematic sampling protocol9

that included the center, midperiphery, and peripheral margin of theoptic nerve in four quadrants (approximately 15% of the total axonnumber). Total axon estimates were calculated by multiplying themean axon density by the total area of the optic nerve. The total opticnerve area was measured by outlining its outer border, and the meanof three area measurements was used. The axon-counting procedurewas performed in a masked fashion without knowledge of the exper-imental status of rat eyes. The percentage axon loss was then ex-pressed by comparing the estimated total number of axons in theocular hypertensive eye relative to the control fellow eye.

Preparation of Retinal Protein Lysates

Retinas were mechanically dissected from enucleated eyes and incu-bated in urea/thiourea lysis buffer (2.5 �L/mg of tissue) containing 9mg/mL dithiothreitol, 40 mg/mL CHAPS (3-[3-cholamidopropyl]di-methylammonio-2-hydroxy-1-propanesulfonate), 0.42 g/mL urea, 0.15g/mL thiourea, 4.45% carrier ampholytes (pH 3–10), and proteaseinhibitors at room temperature for 30 to 60 minutes. The mixture wasthen centrifuged at 15,000g for 5 minutes at 20°C. Protein concentra-tion in the supernatant was measured using a protein assay kit based onthe BCA method (Bio-Rad, Hercules, CA). Protein lysates were imme-diately subjected to the experiments described herein, and all proce-dures were performed at least four times with new protein samples, toensure the reproducibility of results.

Two-Dimensional PolyacrylamideGel Electrophoresis

Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) wasperformed with a Zoom IPGRunner system using 7 cm pH 3–10 NLZoom-Strips and NuAGE Novex 4%–12% Bis-Tris Zoom gels (Invitrogen, Carls-bad, CA). Protein samples were resuspended in IEF buffer (7 M urea, 65mM dithiothreitol, 2% CHAPS, 1% zwittergent, 0.8% ampholytes [pH3–10], 2 M thiourea, and bromophenol blue). For the first dimension,50 to 100 �g of proteins was applied to strips. Isoelectric focusing wasperformed at 175 V for 15 minutes, a 175 to 2000 V ramp for 45minutes, and 2000 V for 30 minutes, at room temperature. For thesecond-dimension separation, the gel strips (ZoomStrips; Invitrogen)were equilibrated for 10 minutes in 45 mM Tris base (pH 7.0) contain-ing 6 M urea, 1.6% SDS, 30% glycerol, and 130 mM dithiothreitol, andthen re-equilibrated for 10 minutes in the same buffer containing 135mM iodoacetamide in place of dithiothreitol. The strips were thenplaced on Bis-Tris gels (ZoomGels; Invitrogen), and unstained molec-ular standards were applied, and electrophoresis was started. Second-dimension gels were run at 200 V/200 mA for 45 minutes. All reagentswere purchased from Genomic Solutions (Ann Arbor, MI).

After electrophoresis, gel slabs were fixed in 10% methanol and 7%acetic acid for 30 minutes at room temperature and then incubatedwith 75 mL of protein gel stain (Sypro Ruby; Bio-Rad) on gentlycontinuous rocker at room temperature overnight. Images were ob-tained using a gel documentation system (Bio-Rad).

Identification of Protein Oxidation

To identify carbonyl groups that are introduced into the amino acidside chain after oxidative modification of proteins, 2D-oxyblot analysiswas performed, as previously described.26 Therefore, the derivativethat is produced by reaction with 2,4-dinitrophenylhydrazine (DNPH)was immunodetected by an antibody specific to the attached DNPmoiety of proteins using a commercial kit (Chemicon, Temecula, CA).Briefly, the electrophoresis was performed in the same way as de-scribed earlier, and the gels were transferred to a nitrocellulose mem-brane using a semidry transfer system (BioRad). Membranes were then

blocked in a buffer (50 mM Tris-HCl, 154 mM NaCl, 0.1% Tween-20[pH 7.5]) containing 5% nonfat dry milk for 1 hour, and incubated witha rabbit anti-DNP antibody (1:150; Chemicon) for 1 hour at roomtemperature. The secondary antibody incubation was performed usinghorseradish peroxidase (HRP)–conjugated anti-rabbit IgG (1:300;Chemicon) for 1 hour at room temperature. The immunoreactivity wasvisualized by enhanced chemiluminescence using commercial re-agents (GE Healthcare, Piscataway, NJ). The kit used for the oxyblotanalysis is sensitive to detect as little as 10 femtomoles of dinitrophenylresidues. To determine specificity, the oxidized proteins provided bythe kit were included as a positive control. Treatment of samples witha control solution served as a negative control to the DNPH treatment.As an additional control, the anti-DNP antibody was omitted.

For protein identification, spots were excised from 2D-gels ob-tained with non–DNPH-treated samples and analyzed by mass spec-trometry. The position of the identified proteins was also confirmed by2D-Western blot analysis using specific antibodies, as previously de-scribed.16,27 Primary antibodies used included monoclonal antibodiesto glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:200; Stress-gen, San Diego, CA), heat shock protein 70-72 (HSP70-72; 1:500;Stressgen), and glutamine synthetase (1:1000; Chemicon). The second-ary antibody was goat anti-mouse IgG conjugated with HRP (1:2000;Sigma-Aldrich, St. Louis, MO).

In-Gel Tryptic Digestion

Samples were prepared using a modification of the technique de-scribed previously.28 After washing of stained gel slabs with 18 M�-cmwater, the selected spots were excised, and gel pieces were incubatedfirst in 0.1 M NH4HCO3 and then in acetonitrile at room temperaturefor 15 minutes. After drying and rehydration steps, gel pieces wereincubated in 20 mM dithiothreitol and 0.1 M NH4HCO3 at 56°C for 45minutes and then in 55 mM iodoacetamide and 0.1 M NH4HCO3 atroom temperature for 30 minutes. These incubations were followed by15-minute incubations in 50 mM NH4HCO3 and acetonitrile. At the endof these series of incubations, gel pieces were dried and then rehy-drated with 20 ng/�L trypsin in 50 mM NH4HCO3. Rehydrated gelpieces were covered with 50 mM NH4HCO3 solution and incubatedovernight at 37°C.

Sample Preparation and Mass Spectrometry

Digests (0.6 �L) were mixed with 0.6 �L �-cyano-4-hydroxy-trans-cinnamic acid (HCCA) saturated in 0.1% trifluoroacetic acid and ace-tonitrile solution (1:1 vol/vol). The mixture was deposited onto a fastevaporation matrix surface obtained by loading 0.5 �L HCCA (10mg/mL in acetone) onto each well of the 96-well target plate. It wasthen washed twice with 2 �L 5% formic acid and analyzed throughmatrix-assisted laser desorption ionization time-of-flight mass spec-trometry (MALDI-TOF/MS; TofSpec 2E mass spectrometer; Micromass,Manchester, UK) in the Reflectron mode. The mass axis was adjustedwith trypsin autolysis peaks (M/z 2239.14, 2211.10, or 842.51) as lockmasses.

In addition, samples were analyzed with liquid chromatography-tandem mass spectrometry (LC/MS/MS). For this purpose, peptides indigested samples were extracted by 15-minute incubations in 5% for-mic acid and acetonitrile. The extracts obtained from repeated pro-cesses were combined and condensed to 1 to 2 �L in a concentrator(SpeedVac; ThermoSavant, Holbrook, NY). Diluted samples were thenanalyzed using a separation system coupled to a mass spectrometer(Q-TOF API-US; Waters, Milford, MA). Briefly, the samples (5 �L) wereinjected onto a 300-�m � 5-mm C18 precolumn (PepMap; LC Packing,Sunnyvale, CA), and after washing, peptides were separated on a75-�m � 150-mm C18 analytical column (Symmetry; Waters) using agradient from 100% solvent A (5% acetonitrile with 0.1% formic acid)to 40% solvent B (95% acetonitrile with 0.1% formic acid) at a flow rateof 200 nL/min for more than 40 minutes. The LC elute was thendirected into the mass spectrometer, and MS/MS spectra were acquired

IOVS, September 2005, Vol. 46, No. 9 Identification of Oxidatively Modified Retinal Proteins in Glaucoma 3179

using the Survey Scan mode. The MS/MS spectra from each ion weresummed and deconvoluted (MaxEnt 3 software; Waters).

Database Search

Bioinformatic databases were searched for protein identification fromtryptic fragment sizes. The Mascot search engine (http://www.matrixscience.com) was initially used by querying the entire theoreti-cal peptide masses in the National Center for Biotechnology Informa-tion (www.ncbi.nlm.nih.gov/ provided in the public domain by thenational Institutes of Health, Bethesda, MD) and SwissProt (http://www.expasy.org/ provided in the public domain by the Swiss Instituteof Bioinformatics, Geneva, Switzerland) protein databases using theassumption that peptides are monoisotopic, may be oxidized at methi-onine residues, and that cysteine is completely modified by iodoacet-amide. Up to one missed trypsin cleavage was allowed. A mass accu-racy tolerance of a maximum of 100 ppm was used for matching thepeptide mass values. Probability-based Mowse scores were obtainedusing the software, by comparison of search results against estimatedrandom match population, and results were reported as �10 � log(P),where P is the absolute probability.29 Scores greater than 71 wereconsidered significant (P � 0.05). We also used the Profound searchengine (http://129.85.19.192/profound_bin/WebProFound.exe) as acomplementary and confirmatory search tool to the former. The z-scores were estimated by comparison of Profound search resultsagainst estimated random match population, which were expressed asdistances to the population mean in units of standard deviation. Scoresgreater than 1.65 were considered significant (P � 0.05).

Analysis of Gel Images

The protein content was determined based on spot intensity in a seriesof 2D-gels obtained using equally loaded protein samples from ocularhypertensive eyes and normotensive fellow eyes in a masked fashion.Coomassie Blue (Bio-Rad)–stained 2D-gels were used to compare pro-tein content, and 2D-oxyblot membranes were used to compare car-bonyl content between ocular hypertensive and control eyes. Aftermatching the corresponding spots on 2D-oxyblot and Coomassie Blue–stained 2D-gel images obtained using the same samples, anti-carbonylreactivity of individual protein spots (the integrated intensity valueafter background subtraction on 2D-oxyblots) was normalized to theirprotein content obtained by measuring the intensity of Coomassie Bluestaining (the integrated intensity value after background subtraction on2D-gels). The percentage change in carbonyl immunoreactivity wasexpressed by comparing the integrated intensities obtained with atleast four different samples from ocular hypertensive eyes relative tothe corresponding control samples. Statistical comparisons were ob-tained using the Mann-Whitney test.

Immunohistochemistry

After fixation, posterior poles of the enucleated rat eyes, includingretinas, were embedded in paraffin and processed for 6-�m longitudi-nal sections, as previously described.13,14,30 To determine the localiza-tion of identified proteins in retinal cell types, histologic sections of theretinas masked for the experimental status of rat eyes were doubleimmunolabeled with specific antibodies, as previously de-scribed.13,14,30 Primary antibodies included monoclonal antibodies toGAPDH (1:200; Stressgen), HSP70-72 (5 �g/mL; Stressgen), and glu-tamine synthetase (1:1000; Chemicon). We used rabbit antibodiesagainst glial fibrillary acidic protein (GFAP), a marker for astrocytes; orbrn-3a, a marker for RGCs (1:400; Chemicon). In addition, 4�,6-dia-midino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eu-gene, OR), a nucleic acid stain, was used to determine intracellulardistribution of immunolabeling. Secondary antibodies were AlexaFluor 488-conjugated anti-mouse or AlexaFluor 568-conjugated anti-rabbit IgG (2 �g/mL; Molecular Probes). The primary antibody waseliminated from the incubation medium, or serum was used to replacethe primary antibody to serve as the negative control. In addition,slides were incubated with each primary antibody followed by the

inappropriate secondary antibody to determine that each secondaryantibody was specific to the species it was made against. Immunola-beled slides were examined using a fluorescence microscope, andimages were recorded by digital photomicrography (Axiovision; CarlZeiss Meditec, Inc.).

Anti-carbonyl reactivity was determined, as previously described.31

Briefly, additional sections were incubated with 0.01% DNPH in 2 NHCl for 1 hour at room temperature. After the washing and blockingsteps, slides were incubated with a monoclonal antibody recognizingDNP (1:100; Zymed, South San Francisco, CA) and then with AlexaFluor 488–conjugated anti-IgG (2 �g/mL; Molecular Probes). Anti-DNPantibody or the DNPH treatment was omitted, or the primary antibodywas replaced with serum to demonstrate immunochemical specificity.

Each specific immunolabeling was performed using at least sixhistologic slides from each eye of at least four experimental rats. Tocontrol variations, histologic slides obtained from ocular hypertensiveand control eyes, as well as the negative controls, were simultaneouslyprocessed during each setting of the immunolabeling.

RESULTS

Proteomic analysis was performed to determine whether reti-nal proteins are oxidatively modified during glaucomatous neu-rodegeneration. Comparison of retinal protein oxidation inocular hypertensive eyes with normotensive fellow eyes wasobtained by identifying proteins containing carbonyl groupsthrough 2D-oxyblot analysis. By evaluating 2D-oxyblots andCoomassie Blue–stained 2D-gels obtained from the same sam-ples, approximately 60 protein spots obtained using retinalprotein lysates from ocular hypertensive eyes (of �400 spots)exhibited protein carbonyl immunoreactivity, which reflectsoxidatively modified proteins (Fig. 2). As shown in Figure 2,positions of proteins on gels obtained using ocular hyperten-sive or control samples were identical. After normalization ofthe anti-DNP reactivity of individual proteins to their contentmeasured on Coomassie Blue–stained 2D-gels, the percentagechange in protein carbonyl immunoreactivity was calculated.There was a significant increase in anti-carbonyl reactivity ofindividual protein spots obtained using retinal protein lysatesfrom ocular hypertensive eyes compared with control eyes(P � 0.01).

However, because the number of RGCs decreases in ocularhypertensive eyes over time, the same amount of retinal pro-tein samples obtained from these eyes may not similarly rep-resent the RGC proteins in the control. To resolve this diffi-culty, we initially attempted to normalize the spot intensity toa RGC marker protein, such as Thy-1.1. However, in accor-dance with previous studies,32 an early decrease was detect-able in Thy-1.1 expression in the retina of ocular hypertensiveeyes before significant neuronal loss after IOP elevation. Ourparallel experiments using immunohistochemistry also demon-strated that glial cells may be positive for many RGC markersduring the experimental paradigm, mostly due to phagocytosisof dying RGCs, which could be another factor interfering withthe prior normalization of spot intensity to an RGC markerprotein. Because of such inconsistencies, the spots exhibitinga new oxidative modification, rather than those exhibiting achange in intensity, were selected for further protein identifi-cation in this initial study. Most important, to determine cellu-lar localization of the identified proteins and retinal distribu-tion of protein oxidation, the findings of proteomic analysiswere further evaluated with immunohistochemistry, as pre-sented later.

Among the oxidized proteins detected on 2D-oxyblots, ap-proximately 20 spots exhibited new protein carbonyls in theretina of ocular hypertensive eyes. To identify those proteinsthat exhibit new oxidative modification, selected spots were


initially analyzed by MALDI-TOF/MS. Because the pretreatmentof proteins with DNPH, which is necessary for immunodetec-tion of reactive carbonyl groups, might affect the results ofmass spectrometric identification of individual proteins, pro-tein spots analyzed by mass spectrometry were excised from2D-gels obtained with non–DNPH-treated protein samples. Asshown in Figure 3, comparison between gel images obtainedusing the same sample, DNPH-treated or -untreated, showed nomajor differences in protein position.

Protein spots (Fig. 2, arrows) were identified through pep-tide mass fingerprinting using two complementary and confir-matory engines for bioinformatic database search. The identi-fied proteins (Fig. 2, numbers 1, 2, and 3) included GAPDH, aglycolytic enzyme; HSP72, a stress protein; and glutamine syn-thetase, an excitotoxicity-related protein, respectively. Massspectra of these proteins and the probability-based Mowsescores obtained with the Mascot search engine are shown inFigure 4. The probability-based Mowse score was 113 (P �0.05) for GAPDH with 46% sequence coverage (number ofpeaks matched, 12); 254 (P � 0.05) for HSP72 with 52%sequence coverage (number of peaks matched, 72); and 131(P � 0.05) for glutamine synthetase with 32% sequence cov-erage (number of peaks matched, 15).

Because the Mascot search engine used for the initial searchqueries all peptide masses regardless of different molecular sizeand isoelectric point of the proteins, the Profound searchengine was also used, by narrowing ranges of the molecularsize and isoelectric point based on positions of protein spotson 2D gels. The Profound search results were consistent withthe Mascot results for all three proteins. The z-scores for allthree proteins were 2.43, with a confidence interval of �99%(P � 0.001), and the sequence coverage was 37%, 56%, and32%; for GAPDH, HSP72, and glutamine synthetase, respec-tively.

Protein identification by MALDI-TOF/MS was subsequentlyconfirmed by peptide sequences obtained by acquiring multi-ple LC/MS/MS spectra for each sample. Figure 5 shows LC/

MS/MS spectra for selected peptides and their sequences,which match with GAPDH, HSP72, and glutamine synthetase.

The molecular weights and isoelectric points estimatedthrough 2D-PAGE matched with the molecular weights and

FIGURE 2. 2D-PAGE of retinal pro-tein lysates. Coomassie Blue-stainedgels and oxyblots were obtained by2D-PAGE of the equally loaded reti-nal protein samples from control orocular hypertensive rat eyes. Proteincarbonyl immunoreactivity detectedon 2D-oxyblots, which reflects oxida-tively modified proteins, occurred toa great extent in the retina of ocularhypertensive eyes. Arrows: proteinspots identified through peptidemass fingerprinting and peptide se-quencing as presented in Figures 4and 5. PI, isoelectric point.

FIGURE 3. 2D-gels stained with Sypro-Ruby (Bio-Rad). Comparisonbetween stained gel images of the same sample, DNPH-untreated (top)and DNPH-treated (bottom) showed no major differences in proteinposition.


isoelectric points of the identified proteins. In addition, theidentified proteins were further confirmed by 2D-Western blotanalysis. The position of proteins identified by peptide massfingerprinting and peptide sequencing matched the results ofimmunodetection on 2D-Western blots using specific antibod-ies for GAPDH, HSP72, and glutamine synthetase (data notshown). Relative percentage changes in the protein carbonylimmunoreactivity of the identified proteins are given in Table1. The increased anti-carbonyl reactivity on 2D-oxyblots ob-tained using retinal protein samples from ocular hypertensiveeyes was significant for all three proteins (P � 0.001).

By determining immunolabeling of tissue sections with spe-cific antibodies, along with an in situ examination of structuralcomponents, immunohistochemistry significantly contributesto protein expression studies using proteomics. Therefore, todetermine cellular localization of the identified proteins, wealso performed immunolabeling of retina sections with specificantibodies. As shown in Figure 6, immunolabeling of retinasections for GAPDH was positive in all cell types, as expected.No difference was detectable in the intensity of GAPDH immu-nolabeling between control (Fig. 6A) and ocular hypertensiveeyes (Fig. 6B). However, whereas the retinal immunolabelingfor GAPDH was mainly cytoplasmic in the control eyes, basedon morphologic assessment of cell types and nuclear DAPIlabeling, many RGCs (but not all) exhibited prominent nuclearimmunolabeling for GAPDH in ocular hypertensive eyes.

Retinal immunolabeling for HSP72 was predominant in theinner retinal layers including both neuronal (GFAP-negativecells) and glial cells (GFAP-positive cells). An increase wasdetectable in the intensity of retinal HSP72 immunolabeling in

ocular hypertensive eyes compared with the controls (Figs.6C, 6D).

Immunolabeling of retina sections for glutamine synthetase,a known marker for Muller cells, demonstrated prominentlabeling of Muller cell bodies located in the inner nuclear layerand their processes contributing to the inner and outer limitingmembranes. Intensity of retinal glutamine synthetase immuno-labeling was similar between control and ocular hypertensiveeyes (Figs. 6E, 6F).

Immunohistochemistry was also used to determine retinaldistribution of the protein carbonyl immunoreactivity. Asshown in Figure 7, immunolabeling of DNPH-treated retinasections with anti-DNP antibody demonstrated that the in-creased anti-carbonyl reactivity in ocular hypertensive eyescompared with the control eyes was detectable mainly in theinner retinal layers. Based on double immunolabeling, proteincarbonyl-positive cells in the inner retinal layers of the ocularhypertensive eyes included brn-3-positive RGCs. This suggeststhat proteins located in the inner retinal layers, including theRGC proteins, are more exposed to oxidative modification inocular hypertensive eyes than those in the outer retinal re-gions.

DISCUSSION

The findings of this in vivo study using a proteomic approachrevealed that protein modification by ROS occurs to a greatextent in the retina of ocular hypertensive eyes. Differentmethods have been used to detect oxidative protein modifica-

FIGURE 4. Peptide mass fingerprinting. The protein spots shown by arrows in Figure 2 were identified by using mass spectrometry andbioinformatics. The identified proteins (Fig. 2, numbers 1, 2, and 3) included a glycolytic enzyme, GAPDH; a stress protein, HSP72; and anexcitotoxicity-related protein, glutamine synthetase, respectively. Left: mass spectra for these spots. Spectral masses (in mass per charge unit, M/z)obtained by MALDI-TOF/MS were analyzed by using bioinformatics through the Mascot and Profound search engines. Right: the probability-basedMowse scores obtained using the Mascot search engine. Among predicted proteins with differential Mowse scores shown as multiple bars on thex-axis, only proteins with Mowse scores greater than 71 (outside the shaded area) were considered significant, which were 113, 254, and 131 (P �0.05) for GAPDH, HSP72, and glutamine synthetase, respectively. The second significant bar indicating a Mowse score of 80 for the samplecorresponding to HSP72 was HSP70, the constitutive form of HSP72. The Profound search results were consistent with the Mascot results, andz-scores for all three proteins were 2.43 (P � 0.001). Asterisks indicate trypsin autolysis peaks.


tions.33 Protein carbonyl formation is a major marker of proteinoxidation, which can arise from direct free radical attack onamino acid side chains. Protein carbonyls react with DNPH,forming a hydrazone that is detectable by immunochemicalmethods34 as used here. Protein oxidation assessed by measur-ing the protein carbonyl content was significantly elevated inthe retina of ocular hypertensive eyes compared with thecontrols.

Several studies have demonstrated that the carbonyl con-tent of proteins increases as a function of aging, a condition in

which proteins are more prone to oxidation.35,36 This chemi-cal modification of proteins has also been implicated in theprogression of neurodegeneration. It is well established thatoxidation is one of the major causes of brain protein damageand dysfunction in several age-related neurodegenerative dis-orders,36,37 including Alzheimer’s disease.26,35,38 In addition,oxidative damage has been associated with retinal injury due toage-related macular degeneration39 or ischemia.40,41 Increasedprotein oxidation in the retina of ocular hypertensive eyesprovides evidence that the oxidative protein damage is alsoassociated with neurodegeneration in glaucoma. Degradationof oxidized proteins by proteases is possible, but oxidativelyinduced, protease-resistant protein cross-linking can occur,thereby preventing this means of removing such proteins.37,42

When not degraded by proteases, oxidatively modified pro-teins can deposit as proteolysis-resistant protein aggregates.37

Accumulation of ineffective oxidized proteins leads to loss ofspecific protein function, abnormal protein clearance, deple-tion of the cellular redox balance, and ultimately cell death.43

Similarly, after oxidative modification of retinal proteins inglaucomatous eyes, the reduced ability of cells to cope with

FIGURE 5. Peptide sequencing. The protein spots shown by arrows in Figure 2 were also analyzed by using liquid chromatography-tandem massspectrometry (LC/MS/MS). This confirmed that the peptide sequences match with GAPDH, HSP72, and glutamine synthetase. LC/MS/MS spectrafor selected peptides and their sequences are shown.

TABLE 1. Relative Percentage Change in Protein CarbonylImmunoreactivity of the Identified Proteins

Identified Protein Specific Oxidation

GAPDH 23 � 4HSP72 12 � 2Glutamine synthetase 51 � 6

Data are presented as the mean � SD. Relative changes in proteincarbonyl immunoreactivity were significant for all three proteins(Mann-Whitney test, P � 0.001).


glaucomatous tissue stress may result in impaired cellular ho-meostasis, eventually contributing to neurodegeneration.

To determine the relationship between retinal protein oxi-dation and glaucomatous neurodegeneration, we identifiedspecific targets of protein oxidation in the retina by coupling2D-oxyblot analysis with peptide mass fingerprinting and pep-tide sequencing. Three specific targets of protein oxidation inthe retina of ocular hypertensive eyes were GAPDH, a glyco-lytic enzyme; HSP72, a stress protein; and glutamine syn-thetase, an excitotoxicity-related protein. Because GAPDH,

HSP72, and glutamine synthetase are known to play importantroles in cell survival and/or function in the retina, their freeradical-mediated modification appears to be associated withthe neurodegenerative process in ocular hypertensive eyes.These are proteins expressed by many cell types through theretina. However, the predominant protein carbonyl immuno-reactivity in the inner retinal layers of the ocular hypertensiveeyes suggests that proteins located in the inner retina, whichprominently include RGC as well as glial cell proteins, are moreexposed to oxidative modification in ocular hypertensive eyes.

FIGURE 6. Immunohistochemicalanalysis of the identified proteins.Retina sections obtained from con-trol and ocular hypertensive rat eyeswere subjected to immunofluores-cence labeling using specific antibod-ies. The merged images presented in(A) and (B) show immunofluores-cence labeling of the retina in con-trol (A) and ocular hypertensive (B)eyes for GAPDH as green, and thenuclear DAPI labeling as blue. RetinalGAPDH immunolabeling included allcell types, as expected, and the in-tensity of GAPDH immunolabelingwas similar in control and ocular hy-pertensive eyes. However, it is nota-ble that many RGCs identified basedon morphologic assessment (arrow-heads) exhibited prominent nuclearimmunolabeling for GAPDH in ocu-lar hypertensive eyes. (C) HSP72 im-munolabeling of a control retina(green) was predominant in the in-ner retinal layers. (D) A similar pat-tern of HSP72 immunolabeling in theretina of an ocular hypertensive eye(green) under higher magnification.The merged image also shows GFAPimmunolabeling. HSP72 immunola-beling was positive in both GFAP-positive astrocytes (yellow) andGFAP-negative neurons (green). TheGFAP-negative neurons in the innerretina are most likely RGCs (arrows).Retinal immunolabeling for glu-tamine synthetase in the control (E)

and ocular hypertensive (F) eyes, which corresponds to Muller cell bodies located in the inner nuclear layer and their processes in the inner andouter limiting membranes. No difference was detectable between the glutamine synthetase immunolabeling of the retina in control and ocularhypertensive eyes. gc, ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar: (A, B, D) 50 �m; (C, E, F) 100 �m.

FIGURE 7. Immunohistochemical analysis of protein carbonyl immunoreactivity. Retina sections obtained from control and ocular hypertensiverat eyes were subjected to immunofluorescence labeling for protein carbonyls. (A, B) Immunofluorescence labeling of DNPH-treated retinalsections with a specific anti-DNP antibody in control and ocular hypertensive eyes, respectively. The increased retinal protein carbonylimmunoreactivity in ocular hypertensive eyes compared with the controls was predominant in the inner retinal layers. (C) Merged image presentedin (B) with another image (not shown) of the same region demonstrating brn-3 immunolabeling. Localization of anti-carbonyl reactivity tobrn-3-positive RGCs (yellow) indicates that RGC proteins are among the retinal proteins exhibiting increased susceptibility to oxidativemodification in ocular hypertensive eyes. Brn-3-negative cells exhibiting protein carbonyl immunoreactivity in the inner nuclear layer are likely theMuller cells. gc, ganglion cell; in, inner nuclear; on, outer nuclear layers. Scale bar, 100 �m.


Although RGCs are preferentially susceptible to glaucomatousinjury, other cell types also prominently respond to wide-spread tissue stress in glaucomatous eyes, which include reti-nal glia.13 This is consistent with the detection of relativelywidespread protein carbonyls in the inner retinal layers ofocular hypertensive eyes. However, the loss in specific proteinfunction due to oxidative modification of the identified pro-teins, separately discussed later in this section, should be par-ticularly important for RGCs, because RGCs are predominantlyinjured in ocular hypertensive eyes and their energetic require-ments are increased to recover from glaucomatous injury andmaintain neuronal survival and function.

One of the proteins demonstrating prominent oxidativemodification in the retina of ocular hypertensive eyes, GAPDH,is a major glycolytic enzyme.44 GAPDH, one of the most abun-dant cellular proteins, has long been known to be easily af-fected by different oxidants, resulting in the loss of dehydro-genase activity. Such oxidant-mediated injury to adenosinediphosphate (ADP) phosphorylation has been shown to resultin impaired energy homeostasis and cell viability.45 Although,mild and reversible oxidation of GAPDH caused by physiolog-ical low concentration of oxidants may initially play a regula-tory role by accelerating glycolysis,46 advanced modification ofthis enzyme has been detrimental.45 Therefore, oxidative inac-tivation of GAPDH may similarly lead to impaired glucoseutilization in the retina of ocular hypertensive eyes. Becausethe survival and function of RGCs are highly energy dependentand glucose is the major substrate for energy metabolism in theretina,47 oxidative modification of GAPDH should be criticallyimportant for RGC survival in glaucoma.

On the other hand, there is now clear evidence that thesignificance of GAPDH is not restricted to its pivotal glycolyticfunction. Besides its metabolic role, GAPDH has been shown tobe involved in many other cellular processes, including DNAand RNA binding and regulation of protein expression.44 Mostimportant, this enzyme plays a role as an apoptosis-signalingprotein48–50 and has been associated with neuronal apoptosisin several neurodegenerative conditions, such as Alzheimer’s,Huntington’s, or Parkinson’s disease.51–53 Nuclear accumula-tion of GAPDH has been associated with the apoptosis signal-ing in neurons48,50,54,55 in conjunction with its functions as aDNA repair enzyme or a nuclear carrier for proapoptotic mol-ecules. The apoptotic role of GAPDH is a consequence of themodification of the GAPDH active site in such a way thatoxidation enhances its binding to nucleic acids.56 Althoughnuclear translocation could facilitate GAPDH functioning inDNA reparation and tRNA transportation during oxidativestress, it has recently been shown that GAPDH can also cleaveRNA.57 These findings suggest that, in addition to its conse-quences leading to impaired energy metabolism, oxidation ofGAPDH could also be a signal for binding with nucleic acidsand changing function. Thus, oxidative modification of GAPDHdetected in ocular hypertensive eyes may be particularly im-portant in the regulation of apoptosis signaling during glauco-matous neurodegeneration through the modification of manyfeatures of this multifunctional protein, including its increasedtranslocation to the nucleus. The prominent nuclear immuno-labeling of many RGCs for GAPDH in ocular hypertensive eyesthat we detected supports this suggestion.

HSP72 was another protein oxidatively modified in theretina of ocular hypertensive eyes. HSPs are molecular chaper-ones involved in many different cellular processes, includingthe folding of newly synthesized proteins, protein traffickingacross cellular membranes, and the assembly/disassembly ofprotein complexes. These proteins are differentially expressedin response to various biological and environmental stresses,indicating that they are involved in the maintenance of cellularfunction and survival.58–60 Obviously, glaucomatous stress

conditions are capable of inducing HSPs, as different HSPs,including HSP27 and HSP60, have been found to be upregu-lated in glaucomatous human donor eyes.10 Overexpression ofHSP72 has also been suggested to play a role as a native defensemechanism in response to tissue stress or injury in glaucoma,in vitro.61 In addition, heat stress or systemic administration ofzinc or geranylgeranylacetone, which induces endogenousHSP expression, has provided neuroprotection in an experi-mental rat model of glaucoma.62,63 Because appropriate ex-pression of HSPs is critical for their function in cellular protec-tion, it is likely that alterations in HSP72 activity due tooxidative protein modification in ocular hypertensive eyescould ultimately lead to the impaired cellular response to tissuestress in these eyes. HSP72 has been identified to protect cellsagainst apoptosis induced by various stress stimuli, includingTNF-�, which has been associated with RGC death in glauco-ma.9,12,16 This function of HSP72 has recently been suggestedto occur mainly upstream of the mitochondria,64 althoughHSP72 is involved in different events associated with the mi-tochondrial cell death pathway.65–67 Regarding the evidenceof mitochondrial dysfunction during RGC death induced byglaucomatous stimuli,9 inactivation of this HSP-associated com-ponent of intrinsic survival signaling could be particularly im-portant in determining the RGC’s fate in glaucoma.

And finally, retinal glutamine synthetase showed prominentprotein carbonyls in ocular hypertensive eyes. Glutamine syn-thetase is a key enzyme that helps maintain the physiologicallevel of extracellular glutamate, thereby modulating excitotox-icity that results from the impaired glutamate–glutamine cycle.Glutamine synthetase has been shown to be particularly sensi-tive to inactivation by oxidants, and altered activity of thisprotein after oxidation has been shown to lead to the accumu-lation of glutamate resulting in neurotoxicity.68 In addition toan age-related decline in glutamine synthetase activity in thehuman brain, oxidatively inactivated glutamine synthetase hasbeen associated with brain injury after ischemia69 or neurode-generative diseases.38,70 Retinal glutamine synthetase, which islocated mainly in the Muller cells,71,72 similarly plays a crucialrole in balancing the neurotoxic effect of glutamate on RGCs.Therefore, our observations in ocular hypertensive eyes sug-gest that after oxidative modification of glutamine synthetase,the resultant failure in the effective conversion of glutamate toglutamine may be associated with the glutamate excitotoxicityto RGCs that has been implicated in the neurodegenerativeprocess of glaucoma. Although multiple efforts did not con-firm73–76 the elevated glutamate levels detected in the vitreousof glaucomatous eyes,77 whether glutamate excitotoxicityplays a role in glaucomatous neurodegeneration still remainselusive. What is also conflicting is that if the intravitreal orintraretinal level of glutamate was elevated in glaucomatouseyes, an increase in the expression of glutamine synthetase byretinal Muller cells would be expected as a compensatorymechanism. However, expression of this protein has not beenfound to be increased in glaucomatous eyes using immunohis-tochemistry.78 Similarly, there was no detectable differencebetween the glutamine synthetase immunolabeling of ocularhypertensive and control retinas in our study. We wonderwhether this might partly be due to oxidative modification ofglutamine synthetase in ocular hypertensive eyes, which mayaffect immunoreactivity by interfering with the antibody bind-ing, and/or may increase the degradation of the oxidized pro-tein. For example, another study using immunochemical de-termination showed that the concentration of glutaminesynthetase significantly decreases after oxidation, indicatingthat the oxidative inactivation leads to the degradation of thisenzyme.79 Thus, although many aspects are yet unclear, alter-ation in glutamine synthetase activity due to oxidative protein


modification appears to be important in controlling the poten-tial neurotoxicity of extracellular glutamate on RGCs.

In summary, the findings of this in vivo study in which weused a proteomic approach provide novel evidence of oxida-tive modification of many retinal proteins in ocular hyperten-sive eyes and identify three specific targets of retinal proteinoxidation. These findings support the association of oxidativedamage with neurodegeneration and the neuroprotective po-tential of antioxidant treatment in glaucoma. This study alsoexemplifies that proteomics is a very promising way for thelarge-scale identification of changes in protein complement,which characterize precise pathogenic mechanisms. By iden-tifying RGC-specific proteome alterations, ongoing studiesshould reveal cellular pathways associated with glaucomatousneurodegeneration at the protein level and provide biomarkersand novel therapeutic targets for neuroprotective interven-tions.

Acknowledgments

The authors thank John B. Klein and William M. Pierce for technicalsupport and valuable discussions.

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Proteomic Identification of Oxidatively Modified Retinal Proteins in a Chronic Pressure-Induced Rat Model of Glaucoma