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KG Shadrach, KA West, M Miyagi, X Gu, RG Salomon, H Sakaguchi, ME Rayborn, JG Hollyfield, JW Crabb; Proteomic Methods for Characterizing Bruch's Membrane . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2787.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop methods for characterizing the age-related changes in Bruch's membrane that lead to thickening and loss of permeability. Bruch's membrane serves as a filter between the retinal pigment epithelium (RPE) and choriocapillaris, influencing the flow of waste products out of and nutrients into the neural retina. Methods: Detergent extraction methods are being developed for recovering Bruch's membrane proteins for identification. Extracted proteins are electrophoresed, gel bands/spots excised and proteins identified by mass spectrometry and sequence database searches. Western and immunocytochemical analyses are used to detect protein oxidative modifications. Results: The protein extraction efficacy has been tested for a high salt buffer (0.5M Tris, 0.2M NaCl, 1%Triton X-100, 1mN PMSF-EDTA), a commercial "Lysis buffer" and an isoelectricfocusing solvent (7M urea, 2M thiourea, 4%CHAPS, 0.5% Triton X100, 2% ampholytes, 1%DTT). Each solution has yielded unique protein components but also common proteins like vitronectin and TIMP-3. The "Lysis buffer" and high salt buffer have provided the greatest number of Coomassie blue detectable SDS-PAGE bands. Bruch's membrane contains proteoglycans and several were identified, such as prolargin, lumincan and bone/cartilage proteoglycan 1. Other detected extracellular matix components include lamin and collagen however many other proteins have been identified. Oxidative protein modifications from docosahexenoate (ie, carboxyethyl pyrrole adducts) and carbohydrate (ie, carboxymethyl lysine ) have also been observed. Conclusion: The Bruch's membrane proteome may be more complex than previously appreciated. Oxidative modifications may be causally involved in age related changes.
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