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Kevin L Schey, Zhen Wang; Quantitative Proteomic Analysis of Lipid-Raft Domains from Lens Fiber Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1682.
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© ARVO (1962-2015); The Authors (2016-present)
Lipid rafts are domains within cell membranes that are rich in cholesterol and sphingolipids and serve to concentrate specific protein activities. Lens fiber cell membranes contain high concentrations of sphingomyelin and cholesterol. Lipid rafts could play critical roles in regulating protein function and in maintaining lens transparency. The purpose of this study is to characterize lens proteins that are localized in raft domains and in non-raft membranes using quantitative proteomic methods.
Bovine lenses were decapsulated and dissected into cortex and nucleus regions. The water-insoluble fraction from each region was divided into two samples and one was treated with methyl-β-cyclodextrin to deplete cholesterol and disrupt rafts. Samples were incubated with detergent (1% Brij 98, 35 mM octyl-glucoside, 600 mM NaCl) at 4 oC for 30 min. and subjected to sucrose density gradient centrifugation. Proteins from low density to high density were isolated and precipitated using chloroform/methanol, digested by trypsin, and analyzed by LC-MS/MS. Raft fractions were identified based on the enrichment of lipid raft markers. Using the iTRAQ quantitative proteomics method, raft proteins were distinguished from non-raft membrane proteins based on abundance changes upon cholesterol-depletion.
Lipid raft fractions were identified as fractions recovered from the interface of the 5% and 35% sucrose layers since raft markers such as flotillin, erlin and prohibitin, were enriched in these fractions and moved to higher density fractions after cholesterol depletion. Additional proteins identified as highly enriched in the raft fractions included caveolins, AQP5, Lim2, and Voltage-dependent calcium channel subunit alpha-2/delta-1. Some proteins were detected in both raft and non-raft fractions including AQP0, neural cell adhesion molecule, Voltage-dependent anion-selective channel proteins, cytoskeleton-associated protein 4, syntaxin, as well as lipid-anchored peripheral membrane proteins such as brain acid soluble protein 1, paralemmin, ras-related C3 botulinum toxin substrate 1, and protein kinase C.
Quantitative proteomic analysis revealed components of the lens fiber cell lipid raft-like, detergent resistant membranes. These data provide clues to protein sorting into distinct membrane microdomains and possibly how protein function is altered depending on the lipid environment.
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