Proteomic analysis of equine amniotic membrane: characterization of proteins

Human amniotic membrane (AM) has been used as a biomaterial for surgical wound skin and ocular surface reconstruction for several years. Currently, equine AM has been used for corneal reconstruction in several animal species, and appears to have the same properties as human AM. Despite the observed positive healing abilities of this tissue in horses with ulcerative keratitis the proteins of equine AM have not been described.

To identify proteins known to be associated with corneal healing from frozen equine AM.

Placentas were acquired from healthy live foal births from a local Thoroughbred breeding farm. The amnion was removed from the chorion by blunt dissection, washed with phosphate-buffered saline (PBS), and treated with 0.05% trypsin and 0.02% ethylene diaminetetraacetic acid in PBS. Amnion was attached to nitrocellulose paper (epithelial side up), and cut into 4 × 4 cm pieces. The sheets were frozen at -80 °C. The protein samples were solubilized, and analyzed by 2D gel electrophoresis and shotgun proteomics.

A reference identification map of the equine AM proteins was produced and 149 different proteins were identified. From gel-based proteomics, 49 spots were excised and 43 proteins identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Shotgun proteomics identified 116 proteins with an overlap of 10 proteins in both analyses.

We have described a reference map for equine AM proteins that may provide a background to explain the positive results found in horses with ulcerative keratopathies using this biomaterial.