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O. Alcazar, A. M. Hawkridge, D. C. Muddiman, S. K. Bhattacharya, M. E. Marin-Castano; Proteomic Characterization of Hydroquinone-Induced Blebs in a Human RPE Cell Line. Invest. Ophthalmol. Vis. Sci. 2008;49(13):484. doi: https://doi.org/.
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Hydroquinone (HQ), a major component of cigarette smoke, which is a risk factor in dry Age-related macular degeneration (AMD), induces formation of non-lethal RPE membrane bleb. This study aims to determine composition of HQ induced RPE bleb proteome, HQ induced bleb glycoproteome, and phosphoproteome in a human RPE cell line.
Human RPE cells were treated with 100 µM HQ for 6 h to allow bleb formation. Blebs were isolated and bleb proteins fractionated on 4-20% gradient gels were assayed for total glycosylation or phosphorylation (GelCode Glycoprotein and Phosphoprotein Staining kits, Pierce; and Pro-Q Diamond and Pro-Q Emerald, Molecular Probes) and subsequently subjected to liquid chromatography tandem mass spectrometry.
Mass spectrometry identified a total of 314 proteins in blebs including glycosylation enzymes, for example, protein O-fucosyltransferase 2. KEGG pathway analysis found identified proteins involved in oxidative phosphorylation, cell junction, and actin cytoskeleton regulation. Glycoprotein and phosphoprotein analyses revealed characteristic patterns in HQ-induced blebs. The blebs showed the presence of several glycoproteins such as phosphatidylinositol glycan class S and Leprecan.
Non-lethal blebs induced with HQ in RPE cells show a characteristic protein profile pattern. Further investigation of these proteins may be useful to identify potential biomarkers and provide insight into the mechanisms leading to dry AMD.
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