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R. Joseph, O. P. Srivastava, J. A. Mobley, R. R. Pfister; Profiling of Epithelial and Stromal Proteins and their Differential Expression in Keratoconus and Normal Human Corneas. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3534.
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The purpose of the study was to identify epithelial and stromal proteins and their up or down regulation in normal and keratoconus human corneas.
The epithelial and stromal proteins from keratoconus and age-matched normal cornea were first analyzed by using Nano-ESI-LC-MS(MS)2 and then by 2D-DIGE (two dimensional-difference gel electrophoresis) methods. During 2D-DIGE analysis, 40 µg epithelial and stromal proteins from keratoconus and normal corneas were labeled with Cy3 and Cy5 dyes, respectively. The Cy2-labeled proteins were used as an internal standard. Similarly, 10 µg of epithelial or stromal proteins from keratoconus and normal corneas were used for Nano-ESI-LC-MS(MS)2 analysis. For identification of protein, the LTQ data was searched through the Transproteomic pipeline equipped with Sequest, Mascot and X! Tandem search engines.
The Nano-ESI-LC-MS(MS)2 analysis identified a total of 95 epithelial proteins from keratoconus and normal corneas. The epithelia of both keratoconus and normal corneas contained keratins 3 and 12 whereas superoxide dismutase was present only in the normal corneas and cathepsin D heavy only in keratoconus corneas. Similarly, a total of 47 stromal proteins were identified in both keratoconus and normal corneas. Collagen alpha-1(VI) and collagen alpha-1(XII) chains were present in the stroma of both keratoconus and normal corneas. The 2D-DIGE analysis showed several post-translationally modified species of cytokeratins 3 and 12 were present in epithelium of keratoconus. Similarly, 2D-DIGE analysis showed differential expression of beta-actin and alpha enolase in stroma of normal and keratoconus corneas.
The results suggest post-translational modification of cytokeratins 3 and12, and the presence of cathepsin D in only the epithelium of keratoconus corneas. Both beta-actin and alpha enolase showed differential expression in stroma of both normal and keratoconus corneas.
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