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DA Ferrington, RJ Kapphahn, JL Louie, EA Peters; Proteomic Analysis of Oxidized Retinal Proteins in Aged Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4533.
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Purpose: It has been proposed that the accumulation of oxidized protein contributes to age-related retinal degeneration. Identification of proteins that are preferentially modified with aging would provide insight into the mechanism behind retinal degeneration. In this study, we identified retinal proteins in aged F344BN rats that were modified by 4-hydroxy-2-nonenal (HNE), a product of lipid peroxidation that inactivates proteins. Methods: To evaluate the extent of oxidative stress and HNE protein modification in the retinas from young and aged rats, we measured the expression of heme-oxygenase 1 (HO-1) and HNE-modified proteins, respectively, by Western immunoblotting. To identify the HNE-modified proteins, retinal proteins were resolved by two-dimensional gel electrophoresis. The HNE-immunopositive proteins were excised from the gel, subjected to in-gel trypsin digestion, and the peptides were analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) and electrospray ionization MS. Results: There was a two-fold upregulation in HO-1 expression and a four-fold increase in HNE-modified proteins in aged retina. Mass spectrometric analysis identified heat shock cognate 70, beta B2 crystallin, and alpha crystallin A as HNE-modified proteins uniquely present in aged retina. Conclusion: These data suggest that the aged retina is under increased oxidative stress, as demonstrated by upregulation of HO-1. Identification of three heat shock proteins containing HNE-modifications suggests that chaperone function may be compromised in the retina with aging. Since chaperones function by preventing protein aggregation and assisting in refolding, the combined effect of increased oxidative stress and diminished chaperone function may place retinal cells from aged organisms at greater risk for oxidative damage.
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