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Hiroaki Kobayashi, Haru Okamoto, Zai-Long Chi, Akira Murakami, Takeshi Iwata; Plasma Proteome Analysis On Cynomolgus Monkey Pedigrees With Early Onset Drusen Formation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):971.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we have reported early onset macular degeneration with drusen in cynomolgus monkey pedigrees (Umeda, et al., IOVS, FASEB J, 2005). These monkeys show similar fundus findings of early stage of age-related maculopathy (ARM) at 2 years after birth. To elucidate mechanism of drusen formation and to find disease biomarkers for early stage of ARM, we performed plasma proteome analysis.
Plasma samples were collected from two affected and control monkeys within the same pedigree. To decrease dynamic range of the protein concentration, plasma samples were first treated with ProteoMiner Large-Capacity Kits (BIO-RAD). The eluted fraction was then separated by 1D SDS-PAGE (12.5% acrylamide gel). Approximately, 65-70 horizontal gel slices were excised by molecular size. Each slice was subjected to treatment with in-gel digestion by trypsin (12.5ng/µl, Sequencing Grade Modified Trypsin, Promega). Tryptic peptides were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS, LCQ DECA XP plus, Thermo Fisher Scientific) and the MS/MS spectra were processed by Bioworks 3.3.1 (Thermo Fisher Scientific) for protein identification.
Successful enrichment of the minor plasma proteins by ProteoMiner Large-Capacity Kits were confirmed by SDS-PAGE. Total of 211 proteins were identified from four samples, including 72 proteins exclusively from the disease group. Six common proteins identified in two disease monkeys were centromere-associated protein E, dermatopontin, gelsolin, HORMA domain-containing protein 1, Ig kappa chain V-I region BAN, and protein Dok-7.
Common proteins specific to disease group would be potentially beneficial as biomarkers for human ARM. One of the identified protein gelsolin is involved with aging process as a regulator of cell structure and metabolism. To study the correlation between identified proteins and drusen formation we are currently analyzing the localization and the function of these protein in the retina.
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