Proteome Analysis of Lipofuscin and Detection of Posttranslational Protein Modifications in Human Retinal Pigment Epithelial Cells
Schütt F.1,2, Überle B.3, Schnölzer M.3, Kopitz J.2, Holz F. G.1
1Department of Ophthalmology, 2Department of Pathobiochemistry, University of Heidelberg
3Protein Analysis Facility, German Cancer Research Center, Heidelberg
Purpose: Lipofuscin accumulation in postmitotic RPE cells is a common downstream pathogenetic pathway in various retinal diseases including age-related macular degeneration. To better understand lipofuscinogenesis and possible adverse effects of its constituents we analyzed the proteome of isolated human lipofuscin granules from human RPE cells and screened for posttranslational modifications.
Method: After homogenization and fractionation by gradient ultracentrifugation of RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Lipofuscin proteins were screened for posttranslational modifications including malondialdehyde (MDA) , 4-hydroxynonenal (HNE) and advanced glycation endproducts (AGE) to identify toxic compounds.
Results: A total of 76 proteins were identified in lipofuscin granules including cytosceleton proteins, proteins of visual cycle, enzymes of metabolism, proteins of mitochondrial respiratory chain, ion channel proteins
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