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L. L. David, P. A. Wilmarth, M. A. Riviere, K. L. Schey, S. Bassnett; Analysis of the Lens Membrane Proteome. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3631.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a sensitive method to identify lens membrane protein components using mass spectrometry.
Three pairs of human lenses (1-day, 4-day, and 8-year old donors) were pooled, and 242 decapsulated lenses from 18-28 day mice were pooled, then each homogenized in phosphate buffer, pellets isolated by centrifugation, extracted twice by brief sonication in 7M urea, followed by a 15 min extraction in 0.1 M NaOH on ice. Membrane material was suspended in n-propanol, reduced with DTT, alkylated with idoacetamide, water added, then pelleted. Following a final delipidation in ethanol, the protein was dissolved in 88% formic acid, diluted to 10% formic acid, and porcine pepsin A added at a enzyme to substrate ratio of 1:50, and incubation performed for 5 hours at 37C. Peptides were solid phase extracted, separated by strong cation exchange into 36 fractions, then analyzed using either capillary or nanospray reverse phase LC-MS/MS with a LTQ linear ion trap (ThermoFinnigan). Tandem MS data were analyzed by Sequest without protease specificity, and a non-redundant, parsimonious list of proteins produced using Scaffold (Proteome Software Inc.). All identified proteins were manually validated.
The methodology identified 164 and 288 proteins in the human and mouse lens membrane samples, respectively. The limited pepsin digestion produced peptide fragments suitable for tandem MS analysis, since an average of 80% of the sequence of aquaporin 0 was identified. The method also allowed relatively specific isolation of membrane components, since the majority of proteins contained transmembrane domains. Many new membrane proteins previously unidentified in lens were observed, including aquaporin 5, which was especially abundant in human lens. Cell adhesion proteins, including N-cadherin and Nr-CAM, were also abundant in both species, as was ras-related C3 botulinum toxin substrate 1.
Identifying the lens membrane proteome is an important first step to understanding its role in lens development, maturation, and cataract formation. The data will aid construction of models of lens membrane architecture.
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