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C. Nordgaard, X. Feng, T.W. Olsen, D.A. Ferrington; Mitochondrial Proteomics of the Retinal Pigment Epithelium at Progressive Stages of Age–Related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1387.
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© ARVO (1962-2015); The Authors (2016-present)
Clinically, alterations in the retinal pigment epithelium (RPE) represent key phenotypic features of age–related macular degeneration (AMD). Recently, we identified changes in the global RPE proteome associated with AMD onset and progression (Nordgaard et al, IOVS, in press). To specifically analyze the subcellular structures implicated in AMD pathogenesis, we isolated a fraction enriched in mitochondria from the RPE of human donor eyes graded using the Minnesota Grading System (MGS).
The RPE was dissected from post–mortem globes graded with the MGS (Olsen & Feng, IOVS, 2004, 45: 4484–90). Differential centrifugation was used to isolate an organelle–rich fraction containing mitochondria, lysosomes, and other subcellular structures. Two–dimensional gel electrophoresis was used to separate organelle proteins in the 3–11 pH range, followed by silver staining and image analysis to identify protein spots that exhibited disease–related expression changes. Spots were then excised for protein identification by MALDI–TOF mass spectrometry.
Differential centrifugation yielded a fraction highly enriched for several organelles implicated in AMD, including mitochondria and lysosomes, as determined by Western blotting for organelle–specific markers. Approximately 320 spots were resolved by 2D gel electrophoresis. Compared to previous analyses of whole–cell lysates, the 2D profile following enrichment was distinctly different. Protein identification by mass spectrometry further verified the presence of organelle–specific proteins, such as mitochondrial heat shock proteins, and that these proteins undergo changes in expression at different disease stages.
Differential centrifugation was successfully combined with proteomics to identify disease–related changes in mitochondrial proteins from the retinal pigment epithelium. The changes in protein expression identified by this study provide insight into the role of distinct subcellular structures in the pathogenic mechanisms underlying AMD and may prove valuable in developing novel therapeutic approaches.
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