Abstract
Purpose: :
Human corneal endothelium (HCEC) is a monolayer of cells whose main purpose is to maintain corneal clarity. Study of HCEC may help us to understand the pathogenesis of corneal endothelial dystrophies such as Fuchs’ Endothelial Dystrophy (FED). So far, Wilson et al compared directly extracted protein from normal corneal endothelium and FED with negative findings. The advance in protein extraction and separation techniques as well as use of proteomic analysis has prompted us to establish HCEC–Descemet’s membrane (HCEC–DM) complex dataset of directly extracted protein. The long term goal is to compare normal to diseased HCEC protein composition.
Methods: :
Two human corneal buttons were recovered from Optisol. HCEC–DM complex was dissected from stroma, placed in ultracentrifuge tube and washed with HEPES buffer at 5,000 rpm, 21 C for 10 minutes prior to extraction. Bio–Rad protein extraction buffer (2 M thiourea, 7 M urea) and tributyl phosphine (TBP) in 100:1 ratio was added to the sample which was mixed by pipetting and ultracentrifuged at 40,000 rpm, 21C for 1 hour. Concentration of samples was determined by modified Bio–Rad protein assay. Equal amounts of protein were loaded onto 17 cm IPG strip (pH range 3–10). Active rehydration was followed by iso–electric focusing. Pre–cast 19 cm gels (8–16% acrylamide, Bio–Rad) were run at 350 V until Bromophenol Blue dye disappeared. Sypro–Ruby staining was performed overnight. Protein spots were imaged by ProXPRESS software (Perkin Elmer TM). Matrix–assisted laser desorption ionization time of flight (MALDI–TOF) was used to identify the spots of interest.
Results: :
Direct protein extraction from a pair of human corneas provided an adequate amount for proteomic identification.Washing of corneas stored in Optisol prior to extraction yielded best protein separation and most reproducible results. We identified 42 spots that had a known match in the general proteomic database. Proteins that have not been identified in HCEC–DM previously were ER–60 protease, prohibitin, peroxiredoxin 2, 3 and 6, ARP3BETA, HNRPH1. Abundant expression of various isoforms of peroxiredoxin implicated a yet undiscovered antioxidant scavenging system required to protect integrity of metabolically active endothelial cells.
Conclusions: :
The established dataset of normally expressed proteins in HCEC–DM could be further employed for comparison to diseased endothelium as seen in FED, with special attention to apoptosis and oxidative stress components of the proteome.
Keywords: cornea: endothelium • proteomics • antioxidants