Abstract
Purpose: :
The purpose of the study was to identify epithelial and stromal proteins and their up or down regulation in normal and keratoconus human corneas.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: keratoconus • proteomics • cornea: basic science