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(Investigative Ophthalmology and Visual Science. 2005;46:3177-3187.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0208
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Proteomic Identification of Oxidatively Modified Retinal Proteins in a Chronic Pressure-Induced Rat Model of Glaucoma

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

1From the Departments of Ophthalmology and Visual Sciences, 2Anatomical Sciences and Neurobiology, and 3Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Based on the evidence of an amplified production of reactive oxygen species (ROS) during glaucomatous neurodegeneration, proteomic analysis was performed to determine oxidative modification of retinal proteins after experimental elevation of intraocular pressure (IOP).

METHODS. IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Protein expression was determined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of retinal protein lysates obtained from eyes matched for the cumulative IOP exposure and axon loss. To determine protein oxidation levels, protein carbonyls were detected through 2D-oxyblot analysis of 2,4-dinitrophenylhydrazine (DNPH)-treated samples using an anti-DNP antibody. For identification of oxidized proteins, peptide masses were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS). In addition to use of different engines in a bioinformatic database search and performance of peptide sequencing and 2D-Western blot analysis for confirmation of the identified proteins, immunohistochemistry was used for further validation of the proteomic findings.

RESULTS. Comparison of 2D-oxyblots with Coomassie Blue–stained 2D-gels revealed that approximately 60 protein spots obtained with retinal protein lysates from ocular hypertensive eyes (of >400 spots) exhibited protein carbonyl immunoreactivity, which reflects oxidatively modified proteins. There was a significant increase in anti-carbonyl reactivity in individual protein spots obtained with retinal protein lysates from ocular hypertensive eyes compared with the control (P < 0.01). The identified proteins through peptide mass fingerprinting and peptide sequencing included glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein. Immunolabeling of retina sections with specific antibodies demonstrated cellular localization of these proteins as well as retinal distribution of the increased protein carbonyl immunoreactivity in ocular hypertensive eyes.

CONCLUSIONS. The findings of this in vivo study provide novel evidence for oxidative modification of many retinal proteins in ocular hypertensive eyes and identify three specific targets of retinal protein oxidation in these eyes, thereby supporting the association of oxidative damage with neurodegeneration in glaucoma. By using a proteomic approach, this study also exemplifies that proteomics provide a very promising way to elucidate pathogenic mechanisms in glaucoma at the protein level.

Mitochondria have been found to be essential in controlling cell death.1 Involvement of mitochondria during apoptosis starts a chain of events that lead to the activation of a proteolytic caspase cascade. In addition, mitochondrial dysfunction results in the compromise of mitochondrial oxidative phosphorylation and production of reactive oxygen species (ROS).2 3 Mitochondrial dysfunction has been associated with neuronal apoptosis in an experimental rat glaucoma model,4 5 and free radical scavenging when combined with trophic factors has provided neuroprotection in ocular hypertensive rat eyes.6 In agreement with these findings, retinal ganglion cells (RGCs) have been shown to be susceptible to ROS,7 and the survival of axotomized neonatal RGCs has been found to be critically sensitive to the oxidative redox state.8 Our recent in vitro studies of primary cultures of RGCs have provided further evidence that the RGC death induced by different glaucomatous stimuli involves both caspase-dependent and caspase-independent components of the mitochondrial cell death pathway, including the generation of ROS. In addition, these in vitro studies revealed that antioxidant treatment improves the survival of caspase-inhibited RGCs by increasing the intrinsic ability of these neurons to survive cytotoxic consequences of mitochondrial dysfunction.9

In addition to alterations identified in the expression level of various proteins in glaucomatous eyes,10 11 12 13 14 it is also evident that many retinal proteins undergo proteolytic cleavage during glaucomatous neurodegeneration15 16 17 or exhibit posttranslational modifications, including phosphorylation.13 18 Novel functions of proteins after such modifications have important implications in the pathogenic mechanisms of glaucoma. Our recent in vitro studies have also demonstrated the inconsistency between gene and protein expression in RGCs and glial cells, mainly due to protein phosphorylation after exposure to glaucomatous stimuli.18 Because the ultimate function of the gene that resides in the protein can be modulated by posttranslational modifications,19 20 studying global changes in the proteome (PROTEins expressed by the genOME) can provide more relevant pathophysiological information.

Based on the evidence demonstrating an amplified production of ROS during glaucomatous neurodegeneration,9 we hypothesized that the "oxidative modification" may be another posttranslational modification of retinal proteins involved in the neurodegenerative process of glaucoma. Given that the identification of alterations in protein complement through proteomic approaches can help to understand impaired cellular mechanisms, we performed proteomic analysis to determine whether retinal proteins are oxidatively modified during glaucomatous neurodegeneration in ocular hypertensive eyes and, if so, what the targets are for protein oxidation in these eyes. Immunochemical detection of protein carbonyls using 2D-oxyblot analysis coupled with peptide mass fingerprinting and peptide sequencing revealed oxidative modification of many retinal proteins in ocular hypertensive eyes and identified three specific targets of protein oxidation in the retinas of these eyes. These novel findings support the association of oxidative damage with neurodegeneration in glaucoma in vivo and exemplify that proteomics provide a very promising way to elucidate pathogenic mechanisms of glaucomatous neurodegeneration at the protein level.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Experimental Rat Glaucoma Model
Intraocular pressure (IOP) elevation was unilaterally induced in male Brown Norway rats with an average weight of 200g by hypertonic saline injections into episcleral veins, as previously described.21 All the animals were handled according to the regulations of the Institutional Animal Care and Use Committee, and all procedures adhered to the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Briefly, in rats under general and topical anesthesia, with placement of a plastic ring around the equator, a glass micropipette (attached to a polyethylene tube connected to a tuberculin syringe) was inserted into a circumferential limbal vein, and approximately 0.1 mL of 1.75 M saline was injected into the venous system. The injection was repeated 1 week later. Unilateral elevation of IOP was induced, and the fellow eyes with normal IOP served as the control. Intraocular pressure was measured in both eyes immediately before and after saline injection, and the measurements were repeated in awake animals22 twice per week using a calibrated hand-held tonometer (Tonopen; Medtronic Solan, Jacksonville, FL). Animals were excluded from the experiments, if there was no measurable IOP exposure, 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 1A demonstrates the course of IOP elevation over time. The percentage axon loss presented in Figure 1B was expressed by comparing the axon count in the ocular hypertensive eye relative to the control fellow eye, as will be described later. The degree of cumulative IOP exposure was estimated by first integrating IOP over time in the ocular hypertensive eye, then subtracting the IOP-time integral from that in the normotensive fellow eye (expressed in units of mm Hg-days), as previously described.23 As shown in Figure 1C , a dose–response effect of pressure on axon loss was detectable with a threshold for axon loss at a value of approximately 200 mm Hg-days. These findings are consistent with previous studies in which the same model was used.21 23 24 Retinas were dissected from the enucleated eyes of animals killed at different time points, and the proteomic experiments described herein were performed with retinal protein lysates obtained from ocular hypertensive and control eyes matched for the cumulative IOP exposure and axon loss. Specifically, the retinal protein samples obtained from moderately damaged eyes with a cumulative IOP exposure of 200 to 400 mm Hg-days were used in this study. As shown in Figure 1 , this level of cumulative IOP exposure corresponds to a relative axon loss of no more than 50%.



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  		FIGURE 1. Experimental rat model of chronic pressure-induced glaucoma. (A, B) The course of IOP elevation and axon loss, respectively, during an experimental period of up to 12 weeks after hypertonic saline injection into the limbal veins in rats. Data are presented as the mean ± SD. The percentage axon loss was determined by comparing the estimated total number of optic nerve axons in the ocular hypertensive eye relative to the control fellow eye. The degree of cumulative IOP exposure was estimated by first integrating IOP over time in the ocular hypertensive eye, then subtracting the IOP-time integral from that in the normotensive fellow eye (mm Hg-days). (C) The association of axon loss with cumulative IOP exposure.

 
Optic Nerve Axon Counts
Optic nerve axons were counted with a protocol similar to that previously used.9 For axon counts, optic nerves excised from enucleated 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. Dehydrated tissues were embedded in epoxy resin, and 1-µm cross sections of the myelinated optic nerves were stained with 1% Toluidine Blue. Captured images were analyzed on computer (Axiovision software; Carl Zeiss Meditec, Inc., Thornwood, NY), as previously described.21 25 Briefly, the axon density was counted in randomized regions of the optic nerve by using a systematic sampling protocol9 that included the center, midperiphery, and peripheral margin of the optic nerve in four quadrants (approximately 15% of the total axon number). Total axon estimates were calculated by multiplying the mean axon density by the total area of the optic nerve. The total optic nerve area was measured by outlining its outer border, and the mean of three area measurements was used. The axon-counting procedure was performed in a masked fashion without knowledge of the experimental status of rat eyes. The percentage axon loss was then expressed by comparing the estimated total number of axons in the ocular hypertensive eye relative to the control fellow eye.

Preparation of Retinal Protein Lysates
Retinas were mechanically dissected from enucleated eyes and incubated in urea/thiourea lysis buffer (2.5 µL/mg of tissue) containing 9 mg/mL dithiothreitol, 40 mg/mL CHAPS (3-[3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate), 0.42 g/mL urea, 0.15 g/mL thiourea, 4.45% carrier ampholytes (pH 3–10), and protease inhibitors at room temperature for 30 to 60 minutes. The mixture was then centrifuged at 15,000g for 5 minutes at 20°C. Protein concentration in the supernatant was measured using a protein assay kit based on the BCA method (Bio-Rad, Hercules, CA). Protein lysates were immediately subjected to the experiments described herein, and all procedures were performed at least four times with new protein samples, to ensure the reproducibility of results.

Two-Dimensional Polyacrylamide Gel Electrophoresis
Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed with a Zoom IPGRunner system using 7 cm pH 3–10 NLZoomStrips and NuAGE Novex 4%–12% Bis-Tris Zoom gels (Invitrogen, Carlsbad, CA). Protein samples were resuspended in IEF buffer (7 M urea, 65 mM dithiothreitol, 2% CHAPS, 1% zwittergent, 0.8% ampholytes [pH 3–10], 2 M thiourea, and bromophenol blue). For the first dimension, 50 to 100 µg of proteins was applied to strips. Isoelectric focusing was performed at 175 V for 15 minutes, a 175 to 2000 V ramp for 45 minutes, and 2000 V for 30 minutes, at room temperature. For the second-dimension separation, the gel strips (ZoomStrips; Invitrogen) were equilibrated for 10 minutes in 45 mM Tris base (pH 7.0) containing 6 M urea, 1.6% SDS, 30% glycerol, and 130 mM dithiothreitol, and then re-equilibrated for 10 minutes in the same buffer containing 135 mM iodoacetamide in place of dithiothreitol. The strips were then placed on Bis-Tris gels (ZoomGels; Invitrogen), and unstained molecular standards were applied, and electrophoresis was started. Second-dimension gels were run at 200 V/200 mA for 45 minutes. All reagents were 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 incubated with 75 mL of protein gel stain (Sypro Ruby; Bio-Rad) on gently continuous rocker at room temperature overnight. Images were obtained using a gel documentation system (Bio-Rad).

Identification of Protein Oxidation
To identify carbonyl groups that are introduced into the amino acid side chain after oxidative modification of proteins, 2D-oxyblot analysis was performed, as previously described.26 Therefore, the derivative that is produced by reaction with 2,4-dinitrophenylhydrazine (DNPH) was immunodetected by an antibody specific to the attached DNP moiety of proteins using a commercial kit (Chemicon, Temecula, CA). Briefly, the electrophoresis was performed in the same way as described earlier, and the gels were transferred to a nitrocellulose membrane 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 with a rabbit anti-DNP antibody (1:150; Chemicon) for 1 hour at room temperature. The secondary antibody incubation was performed using horseradish peroxidase (HRP)–conjugated anti-rabbit IgG (1:300; Chemicon) for 1 hour at room temperature. The immunoreactivity was visualized by enhanced chemiluminescence using commercial reagents (GE Healthcare, Piscataway, NJ). The kit used for the oxyblot analysis is sensitive to detect as little as 10 femtomoles of dinitrophenyl residues. To determine specificity, the oxidized proteins provided by the kit were included as a positive control. Treatment of samples with a 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 obtained with non–DNPH-treated samples and analyzed by mass spectrometry. The position of the identified proteins was also confirmed by 2D-Western blot analysis using specific antibodies, as previously described.16 27 Primary antibodies used included monoclonal antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:200; Stressgen, San Diego, CA), heat shock protein 70-72 (HSP70-72; 1:500; Stressgen), and glutamine synthetase (1:1000; Chemicon). The secondary 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 described previously.28 After washing of stained gel slabs with 18 M{Omega}-cm water, the selected spots were excised, and gel pieces were incubated first in 0.1 M NH4HCO3 and then in acetonitrile at room temperature for 15 minutes. After drying and rehydration steps, gel pieces were incubated in 20 mM dithiothreitol and 0.1 M NH4HCO3 at 56°C for 45 minutes and then in 55 mM iodoacetamide and 0.1 M NH4HCO3 at room temperature for 30 minutes. These incubations were followed by 15-minute incubations in 50 mM NH4HCO3 and acetonitrile. At the end of these series of incubations, gel pieces were dried and then rehydrated with 20 ng/µL trypsin in 50 mM NH4HCO3. Rehydrated gel pieces were covered with 50 mM NH4HCO3 solution and incubated overnight at 37°C.

Sample Preparation and Mass Spectrometry
Digests (0.6 µL) were mixed with 0.6 µL {alpha}-cyano-4-hydroxy-trans-cinnamic acid (HCCA) saturated in 0.1% trifluoroacetic acid and acetonitrile solution (1:1 vol/vol). The mixture was deposited onto a fast evaporation matrix surface obtained by loading 0.5 µL HCCA (10 mg/mL in acetone) onto each well of the 96-well target plate. It was then washed twice with 2 µL 5% formic acid and analyzed through matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS; TofSpec 2E mass spectrometer; Micromass, Manchester, UK) in the Reflectron mode. The mass axis was adjusted with trypsin autolysis peaks (M/z 2239.14, 2211.10, or 842.51) as lock masses.

In addition, samples were analyzed with liquid chromatography-tandem mass spectrometry (LC/MS/MS). For this purpose, peptides in digested samples were extracted by 15-minute incubations in 5% formic acid and acetonitrile. The extracts obtained from repeated processes were combined and condensed to 1 to 2 µL in a concentrator (SpeedVac; ThermoSavant, Holbrook, NY). Diluted samples were then analyzed using a separation system coupled to a mass spectrometer (Q-TOF API-US; Waters, Milford, MA). Briefly, the samples (5 µL) were injected onto a 300-µm x 5-mm C18 precolumn (PepMap; LC Packing, Sunnyvale, CA), and after washing, peptides were separated on a 75-µm x 150-mm C18 analytical column (Symmetry; Waters) using a gradient 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 rate of 200 nL/min for more than 40 minutes. The LC elute was then directed into the mass spectrometer, and MS/MS spectra were acquired using the Survey Scan mode. The MS/MS spectra from each ion were summed and deconvoluted (MaxEnt 3 software; Waters).

Database Search
Bioinformatic databases were searched for protein identification from tryptic fragment sizes. The Mascot search engine (http://www.matrixscience.com) was initially used by querying the entire theoretical peptide masses in the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/ provided in the public domain by the national Institutes of Health, Bethesda, MD) and SwissProt (http://www.expasy.org/ provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland) protein databases using the assumption that peptides are monoisotopic, may be oxidized at methionine residues, and that cysteine is completely modified by iodoacetamide. Up to one missed trypsin cleavage was allowed. A mass accuracy tolerance of a maximum of 100 ppm was used for matching the peptide mass values. Probability-based Mowse scores were obtained using the software, by comparison of search results against estimated random match population, and results were reported as –10 · log(P), where P is the absolute probability.29 Scores greater than 71 were considered significant (P < 0.05). We also used the Profound search engine (http://129.85.19.192/profound_bin/WebProFound.exe) as a complementary and confirmatory search tool to the former. The z-scores were estimated by comparison of Profound search results against estimated random match population, which were expressed as distances to the population mean in units of standard deviation. Scores greater than 1.65 were considered significant (P < 0.05).

Analysis of Gel Images
The protein content was determined based on spot intensity in a series of 2D-gels obtained using equally loaded protein samples from ocular hypertensive eyes and normotensive fellow eyes in a masked fashion. Coomassie Blue (Bio-Rad)–stained 2D-gels were used to compare protein content, and 2D-oxyblot membranes were used to compare carbonyl content between ocular hypertensive and control eyes. After matching the corresponding spots on 2D-oxyblot and Coomassie Blue–stained 2D-gel images obtained using the same samples, anti-carbonyl reactivity of individual protein spots (the integrated intensity value after background subtraction on 2D-oxyblots) was normalized to their protein content obtained by measuring the intensity of Coomassie Blue staining (the integrated intensity value after background subtraction on 2D-gels). The percentage change in carbonyl immunoreactivity was expressed by comparing the integrated intensities obtained with at least four different samples from ocular hypertensive eyes relative to the corresponding control samples. Statistical comparisons were obtained using the Mann-Whitney test.

Immunohistochemistry
After fixation, posterior poles of the enucleated rat eyes, including retinas, were embedded in paraffin and processed for 6-µm longitudinal sections, as previously described.13 14 30 To determine the localization of identified proteins in retinal cell types, histologic sections of the retinas masked for the experimental status of rat eyes were double immunolabeled with specific antibodies, as previously described.13 14 30 Primary antibodies included monoclonal antibodies to GAPDH (1:200; Stressgen), HSP70-72 (5 µg/mL; Stressgen), and glutamine synthetase (1:1000; Chemicon). We used rabbit antibodies against glial fibrillary acidic protein (GFAP), a marker for astrocytes; or brn-3a, a marker for RGCs (1:400; Chemicon). In addition, 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR), a nucleic acid stain, was used to determine intracellular distribution of immunolabeling. Secondary antibodies were Alexa Fluor 488-conjugated anti-mouse or AlexaFluor 568-conjugated anti-rabbit IgG (2 µg/mL; Molecular Probes). The primary antibody was eliminated from the incubation medium, or serum was used to replace the 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 secondary antibody was specific to the species it was made against. Immunolabeled slides were examined using a fluorescence microscope, and images were recorded by digital photomicrography (Axiovision; Carl Zeiss Meditec, Inc.).

Anti-carbonyl reactivity was determined, as previously described.31 Briefly, additional sections were incubated with 0.01% DNPH in 2 N HCl for 1 hour at room temperature. After the washing and blocking steps, slides were incubated with a monoclonal antibody recognizing DNP (1:100; Zymed, South San Francisco, CA) and then with Alexa Fluor 488–conjugated anti-IgG (2 µg/mL; Molecular Probes). Anti-DNP antibody or the DNPH treatment was omitted, or the primary antibody was replaced with serum to demonstrate immunochemical specificity.

Each specific immunolabeling was performed using at least six histologic slides from each eye of at least four experimental rats. To control variations, histologic slides obtained from ocular hypertensive and control eyes, as well as the negative controls, were simultaneously processed during each setting of the immunolabeling.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Proteomic analysis was performed to determine whether retinal proteins are oxidatively modified during glaucomatous neurodegeneration. Comparison of retinal protein oxidation in ocular hypertensive eyes with normotensive fellow eyes was obtained by identifying proteins containing carbonyl groups through 2D-oxyblot analysis. By evaluating 2D-oxyblots and Coomassie Blue–stained 2D-gels obtained from the same samples, approximately 60 protein spots obtained using retinal protein lysates from ocular hypertensive eyes (of >400 spots) exhibited protein carbonyl immunoreactivity, which reflects oxidatively modified proteins (Fig. 2) . As shown in Figure 2 , positions of proteins on gels obtained using ocular hypertensive or control samples were identical. After normalization of the anti-DNP reactivity of individual proteins to their content measured on Coomassie Blue–stained 2D-gels, the percentage change in protein carbonyl immunoreactivity was calculated. There was a significant increase in anti-carbonyl reactivity of individual protein spots obtained using retinal protein lysates from ocular hypertensive eyes compared with control eyes (P < 0.01).



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  		FIGURE 2. 2D-PAGE of retinal protein lysates. Coomassie Blue-stained gels and oxyblots were obtained by 2D-PAGE of the equally loaded retinal protein samples from control or ocular hypertensive rat eyes. Protein carbonyl immunoreactivity detected on 2D-oxyblots, which reflects oxidatively modified proteins, occurred to a great extent in the retina of ocular hypertensive eyes. Arrows: protein spots identified through peptide mass fingerprinting and peptide sequencing as presented in Figures 4 and 5 . PI, isoelectric point.

 
However, because the number of RGCs decreases in ocular hypertensive eyes over time, the same amount of retinal protein samples obtained from these eyes may not similarly represent the RGC proteins in the control. To resolve this difficulty, we initially attempted to normalize the spot intensity to a RGC marker protein, such as Thy-1.1. However, in accordance with previous studies,32 an early decrease was detectable in Thy-1.1 expression in the retina of ocular hypertensive eyes before significant neuronal loss after IOP elevation. Our parallel experiments using immunohistochemistry also demonstrated that glial cells may be positive for many RGC markers during the experimental paradigm, mostly due to phagocytosis of dying RGCs, which could be another factor interfering with the prior normalization of spot intensity to an RGC marker protein. Because of such inconsistencies, the spots exhibiting a new oxidative modification, rather than those exhibiting a change in intensity, were selected for further protein identification in this initial study. Most important, to determine cellular localization of the identified proteins and retinal distribution of protein oxidation, the findings of proteomic analysis were further evaluated with immunohistochemistry, as presented later.

Among the oxidized proteins detected on 2D-oxyblots, approximately 20 spots exhibited new protein carbonyls in the retina of ocular hypertensive eyes. To identify those proteins that exhibit new oxidative modification, selected spots were initially analyzed by MALDI-TOF/MS. Because the pretreatment of proteins with DNPH, which is necessary for immunodetection of reactive carbonyl groups, might affect the results of mass spectrometric identification of individual proteins, protein spots analyzed by mass spectrometry were excised from 2D-gels obtained with non–DNPH-treated protein samples. As shown in Figure 3 , comparison between gel images obtained using the same sample, DNPH-treated or -untreated, showed no major differences in protein position.



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

 
Protein spots (Fig. 2 , arrows) were identified through peptide mass fingerprinting using two complementary and confirmatory engines for bioinformatic database search. The identified proteins (Fig. 2 , numbers 1, 2, and 3) included GAPDH, a glycolytic enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein, respectively. Mass spectra of these proteins and the probability-based Mowse scores obtained with the Mascot search engine are shown in Figure 4 . The probability-based Mowse score was 113 (P < 0.05) for GAPDH with 46% sequence coverage (number of peaks 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 coverage (number of peaks matched, 15).



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  		FIGURE 4. Peptide mass fingerprinting. The protein spots shown by arrows in Figure 2 were identified by using mass spectrometry and bioinformatics. The identified proteins (Fig. 2 , numbers 1, 2, and 3) included a glycolytic enzyme, GAPDH; a stress protein, HSP72; and an excitotoxicity-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-based Mowse scores obtained using the Mascot search engine. Among predicted proteins with differential Mowse scores shown as multiple bars on the x-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 sample corresponding to HSP72 was HSP70, the constitutive form of HSP72. The Profound search results were consistent with the Mascot results, and z-scores for all three proteins were 2.43 (P < 0.001). Asterisks indicate trypsin autolysis peaks.

 
Because the Mascot search engine used for the initial search queries all peptide masses regardless of different molecular size and isoelectric point of the proteins, the Profound search engine was also used, by narrowing ranges of the molecular size and isoelectric point based on positions of protein spots on 2D gels. The Profound search results were consistent with the Mascot results for all three proteins. The z-scores for all three proteins were 2.43, with a confidence interval of >99% (P < 0.001), and the sequence coverage was 37%, 56%, and 32%; for GAPDH, HSP72, and glutamine synthetase, respectively.

Protein identification by MALDI-TOF/MS was subsequently confirmed by peptide sequences obtained by acquiring multiple 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.



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

 
The molecular weights and isoelectric points estimated through 2D-PAGE matched with the molecular weights and isoelectric points of the identified proteins. In addition, the identified proteins were further confirmed by 2D-Western blot analysis. The position of proteins identified by peptide mass fingerprinting and peptide sequencing matched the results of immunodetection on 2D-Western blots using specific antibodies for GAPDH, HSP72, and glutamine synthetase (data not shown). Relative percentage changes in the protein carbonyl immunoreactivity of the identified proteins are given in Table 1 . The increased anti-carbonyl reactivity on 2D-oxyblots obtained using retinal protein samples from ocular hypertensive eyes was significant for all three proteins (P < 0.001).


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

 
By determining immunolabeling of tissue sections with specific antibodies, along with an in situ examination of structural components, immunohistochemistry significantly contributes to protein expression studies using proteomics. Therefore, to determine cellular localization of the identified proteins, we also performed immunolabeling of retina sections with specific antibodies. As shown in Figure 6 , immunolabeling of retina sections for GAPDH was positive in all cell types, as expected. No difference was detectable in the intensity of GAPDH immunolabeling between control (Fig. 6A) and ocular hypertensive eyes (Fig. 6B) . However, whereas the retinal immunolabeling for GAPDH was mainly cytoplasmic in the control eyes, based on morphologic assessment of cell types and nuclear DAPI labeling, many RGCs (but not all) exhibited prominent nuclear immunolabeling for GAPDH in ocular hypertensive eyes.



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  		FIGURE 6. Immunohistochemical analysis of the identified proteins. Retina sections obtained from control and ocular hypertensive rat eyes were subjected to immunofluorescence labeling using specific antibodies. The merged images presented in (A) and (B) show immunofluorescence labeling of the retina in control (A) and ocular hypertensive (B) eyes for GAPDH as green, and the nuclear DAPI labeling as blue. Retinal GAPDH immunolabeling included all cell types, as expected, and the intensity of GAPDH immunolabeling was similar in control and ocular hypertensive eyes. However, it is notable that many RGCs identified based on morphologic assessment (arrowheads) exhibited prominent nuclear immunolabeling for GAPDH in ocular hypertensive eyes. (C) HSP72 immunolabeling of a control retina (green) was predominant in the inner retinal layers. (D) A similar pattern of HSP72 immunolabeling in the retina of an ocular hypertensive eye (green) under higher magnification. The merged image also shows GFAP immunolabeling. HSP72 immunolabeling was positive in both GFAP-positive astrocytes (yellow) and GFAP-negative neurons (green). The GFAP-negative neurons in the inner retina are most likely RGCs (arrows). Retinal immunolabeling for glutamine synthetase in the control (E) and ocular hypertensive (F) eyes, which corresponds to Müller cell bodies located in the inner nuclear layer and their processes in the inner and outer limiting membranes. No difference was detectable between the glutamine synthetase immunolabeling of the retina in control and ocular hypertensive eyes. gc, ganglion cell layer; in, inner nuclear layer; on, outer nuclear layer. Scale bar: (A, B, D) 50 µm; (C, E, F) 100 µm.

 
Retinal immunolabeling for HSP72 was predominant in the inner retinal layers including both neuronal (GFAP-negative cells) and glial cells (GFAP-positive cells). An increase was detectable 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 Müller cells, demonstrated prominent labeling of Müller cell bodies located in the inner nuclear layer and their processes contributing to the inner and outer limiting membranes. Intensity of retinal glutamine synthetase immunolabeling was similar between control and ocular hypertensive eyes (Figs. 6E 6F) .

Immunohistochemistry was also used to determine retinal distribution of the protein carbonyl immunoreactivity. As shown in Figure 7 , immunolabeling of DNPH-treated retina sections with anti-DNP antibody demonstrated that the increased anti-carbonyl reactivity in ocular hypertensive eyes compared with the control eyes was detectable mainly in the inner retinal layers. Based on double immunolabeling, protein carbonyl-positive cells in the inner retinal layers of the ocular hypertensive eyes included brn-3-positive RGCs. This suggests that proteins located in the inner retinal layers, including the RGC proteins, are more exposed to oxidative modification in ocular hypertensive eyes than those in the outer retinal regions.



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

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The findings of this in vivo study using a proteomic approach revealed that protein modification by ROS occurs to a great extent in the retina of ocular hypertensive eyes. Different methods have been used to detect oxidative protein modifications.33 Protein carbonyl formation is a major marker of protein oxidation, which can arise from direct free radical attack on amino acid side chains. Protein carbonyls react with DNPH, forming a hydrazone that is detectable by immunochemical methods34 as used here. Protein oxidation assessed by measuring the protein carbonyl content was significantly elevated in the retina of ocular hypertensive eyes compared with the controls.

Several studies have demonstrated that the carbonyl content of proteins increases as a function of aging, a condition in which proteins are more prone to oxidation.35 36 This chemical modification of proteins has also been implicated in the progression of neurodegeneration. It is well established that oxidation is one of the major causes of brain protein damage and dysfunction in several age-related neurodegenerative disorders,36 37 including Alzheimer’s disease.26 35 38 In addition, oxidative damage has been associated with retinal injury due to age-related macular degeneration39 or ischemia.40 41 Increased protein oxidation in the retina of ocular hypertensive eyes provides evidence that the oxidative protein damage is also associated with neurodegeneration in glaucoma. Degradation of oxidized proteins by proteases is possible, but oxidatively induced, protease-resistant protein cross-linking can occur, thereby preventing this means of removing such proteins.37 42 When not degraded by proteases, oxidatively modified proteins can deposit as proteolysis-resistant protein aggregates.37 Accumulation of ineffective oxidized proteins leads to loss of specific protein function, abnormal protein clearance, depletion of the cellular redox balance, and ultimately cell death.43 Similarly, after oxidative modification of retinal proteins in glaucomatous eyes, the reduced ability of cells to cope with glaucomatous tissue stress may result in impaired cellular homeostasis, eventually contributing to neurodegeneration.

To determine the relationship between retinal protein oxidation and glaucomatous neurodegeneration, we identified specific targets of protein oxidation in the retina by coupling 2D-oxyblot analysis with peptide mass fingerprinting and peptide sequencing. Three specific targets of protein oxidation in the retina of ocular hypertensive eyes were GAPDH, a glycolytic enzyme; HSP72, a stress protein; and glutamine synthetase, an excitotoxicity-related protein. Because GAPDH, HSP72, and glutamine synthetase are known to play important roles in cell survival and/or function in the retina, their free radical-mediated modification appears to be associated with the neurodegenerative process in ocular hypertensive eyes. These are proteins expressed by many cell types through the retina. However, the predominant protein carbonyl immunoreactivity in the inner retinal layers of the ocular hypertensive eyes suggests that proteins located in the inner retina, which prominently include RGC as well as glial cell proteins, are more exposed to oxidative modification in ocular hypertensive eyes. Although RGCs are preferentially susceptible to glaucomatous injury, other cell types also prominently respond to widespread tissue stress in glaucomatous eyes, which include retinal glia.13 This is consistent with the detection of relatively widespread protein carbonyls in the inner retinal layers of ocular hypertensive eyes. However, the loss in specific protein function due to oxidative modification of the identified proteins, separately discussed later in this section, should be particularly important for RGCs, because RGCs are predominantly injured in ocular hypertensive eyes and their energetic requirements are increased to recover from glaucomatous injury and maintain neuronal survival and function.

One of the proteins demonstrating prominent oxidative modification in the retina of ocular hypertensive eyes, GAPDH, is a major glycolytic enzyme.44 GAPDH, one of the most abundant cellular proteins, has long been known to be easily affected by different oxidants, resulting in the loss of dehydrogenase activity. Such oxidant-mediated injury to adenosine diphosphate (ADP) phosphorylation has been shown to result in impaired energy homeostasis and cell viability.45 Although, mild and reversible oxidation of GAPDH caused by physiological low concentration of oxidants may initially play a regulatory role by accelerating glycolysis,46 advanced modification of this enzyme has been detrimental.45 Therefore, oxidative inactivation of GAPDH may similarly lead to impaired glucose utilization in the retina of ocular hypertensive eyes. Because the survival and function of RGCs are highly energy dependent and glucose is the major substrate for energy metabolism in the retina,47 oxidative modification of GAPDH should be critically important for RGC survival in glaucoma.

On the other hand, there is now clear evidence that the significance of GAPDH is not restricted to its pivotal glycolytic function. Besides its metabolic role, GAPDH has been shown to be involved in many other cellular processes, including DNA and RNA binding and regulation of protein expression.44 Most important, this enzyme plays a role as an apoptosis-signaling protein48 49 50 and has been associated with neuronal apoptosis in several neurodegenerative conditions, such as Alzheimer’s, Huntington’s, or Parkinson’s disease.51 52 53 Nuclear accumulation of GAPDH has been associated with the apoptosis signaling in neurons48 50 54 55 in conjunction with its functions as a DNA repair enzyme or a nuclear carrier for proapoptotic molecules. The apoptotic role of GAPDH is a consequence of the modification of the GAPDH active site in such a way that oxidation enhances its binding to nucleic acids.56 Although nuclear translocation could facilitate GAPDH functioning in DNA reparation and tRNA transportation during oxidative stress, it has recently been shown that GAPDH can also cleave RNA.57 These findings suggest that, in addition to its consequences leading to impaired energy metabolism, oxidation of GAPDH could also be a signal for binding with nucleic acids and changing function. Thus, oxidative modification of GAPDH detected in ocular hypertensive eyes may be particularly important in the regulation of apoptosis signaling during glaucomatous neurodegeneration through the modification of many features of this multifunctional protein, including its increased translocation to the nucleus. The prominent nuclear immunolabeling of many RGCs for GAPDH in ocular hypertensive eyes that we detected supports this suggestion.

HSP72 was another protein oxidatively modified in the retina of ocular hypertensive eyes. HSPs are molecular chaperones involved in many different cellular processes, including the folding of newly synthesized proteins, protein trafficking across cellular membranes, and the assembly/disassembly of protein complexes. These proteins are differentially expressed in response to various biological and environmental stresses, indicating that they are involved in the maintenance of cellular function and survival.58 59 60 Obviously, glaucomatous stress conditions are capable of inducing HSPs, as different HSPs, including HSP27 and HSP60, have been found to be upregulated in glaucomatous human donor eyes.10 Overexpression of HSP72 has also been suggested to play a role as a native defense mechanism in response to tissue stress or injury in glaucoma, in vitro.61 In addition, heat stress or systemic administration of zinc or geranylgeranylacetone, which induces endogenous HSP expression, has provided neuroprotection in an experimental rat model of glaucoma.62 63 Because appropriate expression of HSPs is critical for their function in cellular protection, it is likely that alterations in HSP72 activity due to oxidative protein modification in ocular hypertensive eyes could ultimately lead to the impaired cellular response to tissue stress in these eyes. HSP72 has been identified to protect cells against apoptosis induced by various stress stimuli, including TNF-{alpha}, which has been associated with RGC death in glaucoma.9 12 16 This function of HSP72 has recently been suggested to occur mainly upstream of the mitochondria,64 although HSP72 is involved in different events associated with the mitochondrial cell death pathway.65 66 67 Regarding the evidence of mitochondrial dysfunction during RGC death induced by glaucomatous stimuli,9 inactivation of this HSP-associated component of intrinsic survival signaling could be particularly important in determining the RGC’s fate in glaucoma.

And finally, retinal glutamine synthetase showed prominent protein carbonyls in ocular hypertensive eyes. Glutamine synthetase is a key enzyme that helps maintain the physiological level of extracellular glutamate, thereby modulating excitotoxicity that results from the impaired glutamate–glutamine cycle. Glutamine synthetase has been shown to be particularly sensitive to inactivation by oxidants, and altered activity of this protein after oxidation has been shown to lead to the accumulation of glutamate resulting in neurotoxicity.68 In addition to an age-related decline in glutamine synthetase activity in the human brain, oxidatively inactivated glutamine synthetase has been associated with brain injury after ischemia69 or neurodegenerative diseases.38 70 Retinal glutamine synthetase, which is located mainly in the Müller cells,71 72 similarly plays a crucial role in balancing the neurotoxic effect of glutamate on RGCs. Therefore, our observations in ocular hypertensive eyes suggest that after oxidative modification of glutamine synthetase, the resultant failure in the effective conversion of glutamate to glutamine may be associated with the glutamate excitotoxicity to RGCs that has been implicated in the neurodegenerative process of glaucoma. Although multiple efforts did not confirm73 74 75 76 the elevated glutamate levels detected in the vitreous of glaucomatous eyes,77 whether glutamate excitotoxicity plays a role in glaucomatous neurodegeneration still remains elusive. What is also conflicting is that if the intravitreal or intraretinal level of glutamate was elevated in glaucomatous eyes, an increase in the expression of glutamine synthetase by retinal Müller cells would be expected as a compensatory mechanism. However, expression of this protein has not been found to be increased in glaucomatous eyes using immunohistochemistry.78 Similarly, there was no detectable difference between the glutamine synthetase immunolabeling of ocular hypertensive and control retinas in our study. We wonder whether this might partly be due to oxidative modification of glutamine synthetase in ocular hypertensive eyes, which may affect immunoreactivity by interfering with the antibody binding, and/or may increase the degradation of the oxidized protein. For example, another study using immunochemical determination showed that the concentration of glutamine synthetase significantly decreases after oxidation, indicating that the oxidative inactivation leads to the degradation of this enzyme.79 Thus, although many aspects are yet unclear, alteration in glutamine synthetase activity due to oxidative protein modification appears to be important in controlling the potential neurotoxicity of extracellular glutamate on RGCs.

In summary, the findings of this in vivo study in which we used a proteomic approach provide novel evidence of oxidative modification of many retinal proteins in ocular hypertensive eyes and identify three specific targets of retinal protein oxidation. These findings support the association of oxidative damage with neurodegeneration and the neuroprotective potential of antioxidant treatment in glaucoma. This study also exemplifies that proteomics is a very promising way for the large-scale identification of changes in protein complement, which characterize precise pathogenic mechanisms. By identifying RGC-specific proteome alterations, ongoing studies should reveal cellular pathways associated with glaucomatous neurodegeneration at the protein level and provide biomarkers and novel therapeutic targets for neuroprotective interventions.


   Acknowledgements
 
The authors thank John B. Klein and William M. Pierce for technical support and valuable discussions.


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

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

Disclosure: G. Tezel, None; X. Yang, None; J. Cai, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Gülgün Tezel, University of Louisville School of Medicine, Kentucky Lions Eye Center, 301 E. Muhammad Ali Boulevard, Louisville, KY 40202; gulgun.tezel{at}louisville.edu.


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