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M. Shibuya, Y. Tanaka, V.N. Reddy, J. Utsumi, T. Iwata; Differential Proteome Analysis by Isotope Ethanol Tagging of Proteins Extracted From RPE Cells Treated With Retinal Pigment Epithelial Cells Factor–1 (REF–1) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5136.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously reported a growth promotive factor REF–1, which specifically stimulate the proliferation of human RPE cells (IOVS 45:245–252, 2004). To investigate the change of protein expression in human RPE cells by REF–1 treatment, proteome analysis using ion trap LC–MS/MS was performed. Methods: The REF–1 treated and non–treated human primary RPE cells were grown in DMEM medium with 15% FCS for 24, 48 and 72 hours. Proteins extracted from human RPE cells were treated with trypsin for 6h at 37 degree. REF–1 treated and non–treated proteins cell extract were separately labeled with distinct stable–isotope ethanol tag (ethanol containing 1H/2H). After combining the two, the sample was separated by multiple dimension liquid chromatography and analyzed by ion spray tandem mass spectrometer (Finnigan LCQ DECA XP plus, Thermo Electron Corporation). The data analysis software (Bioworks v3.1, Thermo Electron) was used to identify the proteins. Results: One hundred ninety five proteins were labeled with tag and measured in the REF–1 treated and non–treated samples. One hundred thirty eight proteins were increase and 47 proteins were decrease. ATP–binding cassette protein, SEC8, REC8, and CDC14 were increase by 4.5, 3.8, 3.8, and 2.3–fold, respectively by REF–1–treatment. Coagulation Factor V, Chondroitin sulfate, and proteoglycan 6 were decrease by 14.3, 14.3, and 16.7–fold, respectively by REF–1 treatment. Conclusions: A new method was applied to measure differential protein expression of RPE cells treated with REF–1. The method reproducibility was confirmed and the new tags and analysis methods allowed peptides from different samples to be identified by their relative abundance ratio with greater ease and accuracy than other methods. Detail data of proteins will be presented at the poster.
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