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Vasanth Rao, Rupalatha Maddala, Nikolai Skiba; Proteomic Analysis of Trabecular Meshwork Extracellular Matrix. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3533.
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© ARVO (1962-2015); The Authors (2016-present)
The role of extracellular matrix (ECM) synthesis, turnover and organization in influencing aqueous humor outflow through the trabecular pathway and intraocular pressure, has been heavily contested. However, there is not much by way of a comprehensive analysis of trabecular meshwork (TM) tissue ECM proteins in the literature. Here we provide the ECM protein composition of porcine TM tissue analyzed by Synapt G2 mass spectrometry.
TM tissue extracted from freshly enucleated porcine eyes was minced and digested with dispase, and homogenized in high salt Tris buffer solution. The 7000 g pellet derived from the tissue homogenate was dissolved in 2 M urea buffer and stirred overnight. The 14,000 g supernatant from urea dissolved tissue specimens was separated by gradient SDS-PAGE (4-15% acrylamide) and proteins stained using GelCode blue. Distinct protein bands from SDS-PAGE gels were extracted, digested in-gel with trypsin and identified by mass spectrometric analysis using a Synapt G2 Water system.
The procedure described above produced consistent results (n=4) with respect to extracting predominantly (>80%) ECM proteins from the TM tissue. Additionally, Synapt G2 mass spectrometry-based proteomics analysis of the ECM enriched fraction from TM tissue revealed the presence of Perlecan, Collagen α1, Collagen α3, Prolargin, Biglycan, Decorin, Fibromodulin, Thrombospondin-1, EGF-containing fubulin-1, Mimecan, sushi-repeat- containing proteins (SRPX1 and 2), Fibulin-5, Fibulin-1, C1q/TNF-related proteins, Lumican, Fibronectin, Laminin, Type VI collagen, Galectin-1, Matrillin-2, Tenascin XB, Nidogen-2, Periostin, Podocan and other ECM and ECM -associated proteins as some of the predominant proteins. Based on these reproducible observations, the effects of elevated intraocular pressure (50 mm Hg) on the ECM composition of porcine TM tissue are being assessed by quantitative proteomics analysis to identify alterations in specific ECM proteins.
This study provides a reproducible protocol for the extraction of ECM proteins from TM tissue and comprehensive data on the identification and composition of ECM proteins in the TM tissue, as evaluated using proteomics analysis.
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