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B.G. Gugiu, M. Rozanowska, B. Rozanowski, M.E. Rayborn, V.L. Bonilha, X. Gu, R.G. Salomon, J.G. Hollyfield, M.E. Boulton, J.W. Crabb; Proteomic and Ultrastructural Analyses of Human Lipofuscin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1739.
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Purpose: The progressive accumulation of lipofuscin in the retinal pigment epithelium (RPE), correlates with the pathogenesis of age–related macular degeneration (AMD). We seek a better molecular understanding of the sources and consequences of lipofuscin accumulation, including the protein content of lipofuscin. Methods: Human RPE lipofuscin was purified by conventional sucrose density gradient centrifugation methods. Lipofuscin granule purity was evaluated by light, fluorescence, confocal, and electron microscopy. Lipofuscin preparations were extracted with chloroform/methanol then the chloroform insoluble material was extracted with SDS and subjected to SDS–PAGE, gel bands excised and proteins identified by LC MS/MS. Western analysis was used to probe for oxidative protein modifications. Results: Ultrastructural analyses of lipofuscin purified by conventional methods revealed a heterogeneous core structure composed of lipofuscin granules surrounded by substantial extra–granular material. The chloroform insoluble lipofuscin fraction of the conventional preparation exhibited many fuzzy Coomassie blue stained SDS–PAGE bands, suggesting post–translational modifications. Western blot analysis confirmed the presence of abundant carboxyethylpyrrole adducts. Over 160 proteins were identified, ∼33% of which exhibited apparent mass additions. Essentially "pure" lipofuscin granules, free of extra–granular material, were obtained by proteolytic digestion of the conventional preparation. Boiling the purified granules in SDS has so far failed to yield SDS–PAGE detectable bands with Coomassie or silver staining. Conclusions: Lipofuscin granules appear to be embedded in a protein "matrix" similar in content to drusen. Proteomic characterization of purified lipofuscin granules is underway. CR: N. Supported in part by NIH grants EY06603, EY14239, EY14240, EY15638, GM21249, HL53315, Fight for Sight, The Foundation Fighting Blindness, The Cleveland Clinic Foundation and The Wellcome Trust, UK.
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