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G. Tezel, X. Yang, J. Cai; Proteomic Identification of Oxidatively Modified Retinal Proteins in a Chronic Pressure–Induced Rat Model of Glaucoma . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4729.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Based on the evidence of an amplified production of ROS during glaucomatous neurodegeneration, to establish a relationship between protein oxidation and neurodegeneration, proteomic analysis was performed to identify oxidatively modified retinal proteins following chronic elevation of IOP. Methods: IOP elevation was induced in rats by saline injections into limbal veins. Retinal protein expression was determined by 2D PAGE in eyes matched for the cumulative IOP exposure (IOP–time integral) and axon loss. Protein oxidation levels were compared using 2D Oxyblot analysis. For protein identification, spots were excised from Sypro Ruby–stained gels obtained without DNPH treatment. Peptide masses were identified by MALDI–TOF mass spectrometry and bioinformatics. In addition to using different search engines for peptide mass fingerprinting, 2D Western blot analysis and immunohistochemistry were utilized for further confirmation. Results: Comparison of 2D Oxyblots with Coomassie Blue–stained 2D gels from the same samples revealed that approximately 60 protein spots obtained using protein lysates from hypertensive retinas (out of >300 spots) exhibited anti–protein carbonyl immunoreactivity, which reflects oxidatively modified proteins. There was a significant increase in anti–carbonyl immunoreactivity in individual protein spots obtained using lysates from hypertensive retinas compared with the controls (p<0.01). The identified proteins included a glycolytic enzyme, GAPDH; a stress protein, hsp72; and an excitotoxicity–related protein, glutamine synthetase. 2D Western blots confirmed the identified proteins, and immunolabeling of retina sections with specific antibodies demonstrated their cellular localization and the retinal pattern of anti–carbonyl reactivity. Conclusions: Findings of this study revealed that protein modification by ROS occurs to a great extent in hypertensive retinas. By identifying the specific targets of protein oxidation, this study supports the association of oxidative damage with neurodegeneration in glaucoma, since oxidatively modified proteins may lead to the loss in specific protein function, abnormal protein clearance, depletion of the cellular redox balance, and ultimate cell death. By presenting the first proteomic approach, this study also exemplifies that proteomics provide a very promising tool to elucidate pathogenic mechanisms in glaucoma at protein level.
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