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A. M. Dins, C. J. Lieven, J. D. Ribich, L. A. Levin; Redox Proteomic Identification of Molecules Undergoing Disulfide Formation as a Result of Experimental Retinal Ischemia. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1400.
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Retinal ischemia is a common result of embolic disease, diabetic vasculopathy, venous disease, and very high elevations of intraocular pressure (IOP). Ischemia followed by reflow causes production of certain reactive oxygen species (ROS), which both cause direct cellular injury and may signal apoptosis. To elucidate the latter signaling pathways, we used a redox proteomic approach to identify molecules undergoing formation of intermolecular disulfide bonds as a result of acute retinal ischemia.
The ischemia model employed raising the IOP to 110 mmHg in one eye of anesthetized Long Evans rats for 60 min. The contralateral eyes served as controls. Retinas were removed immediately or 4 hours after ischemia and prepared for gel electrophoresis. Following a first dimension run in non-reducing conditions, the gel lanes were excised and incubated in dithiothreitol (DTT) to reduce disulfide bonds, followed by iodoacetamide to alkylate free sulfhydryls. After this incubation the gel lanes were rotated 90 degrees and run in the second dimension. Gels were stained with SYPRO Ruby and individual spots differing between conditions identified for subsequent mass spectrometry.
The vast majority of the proteins in the second dimension were on a 45 degree diagonal, indicating that they did not contain disulfide bonds. Several spots appeared below the diagonal, indicating the presence of disulfides that had been reduced during the DTT incubation. There were a small number of protein spots that were present in the 4 hour ischemia model and not in the control or 0 hour ischemia model, indicating the presence of ischemia-dependent formation of intermolecular disulfide bonds.
Proteins that undergo disulfide formation after ischemia may be important ROS-dependent signaling molecules for retinal neuronal death. These proteins may be novel therapeutic targets for intervention in diseases characterized by retinal ischemia.
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