Proteomic analysis of equine amniotic membrane: characterization of proteins.

The research paper presents a study that aimed to identify and create a reference map of proteins residing in the equine amniotic membrane (AM), which demonstrate properties similar to human AM and are beneficial in corneal healing and reconstruction.

Objective and Methodology

The study’s objective was to identify the proteins present in the equine AM, especially those associated with corneal healing. The researchers collected placentas from live foal births from a local Thoroughbred breeding farm. They separated the amnion from the chorion through blunt dissection and subjected it to a series of processes including washing with phosphate-buffered saline, treating with trypsin and ethylenediaminetetraacetic acid, and attaching it to nitrocellulose paper.

• The amnion was then cut and frozen at very low temperatures (-80 °C).
• The frozen amnion was then used for protein isolation and subjected to 2D gel electrophoresis and a process known as shotgun proteomics for protein identification.

Results of the Study

• A reference map, which cataloged the proteins in the equine AM, was created after the analysis and 149 distinct proteins were identified.
• The use of gel-based proteomics lead to excision of 49 spots with 43 proteins subsequently identified through the use of liquid chromatography and tandem mass spectrometry (LC-MS/MS).
• The shotgun proteomics method identified a total of 116 proteins, with an overlap of 10 proteins found in both the gel-based and shotgun methods.

Conclusion

The successful mapping and identification of proteins within the equine AM performed by this study provides context for the positive results achieved in horses dealing with ulcerative keratopathies that made use of this biomaterial. In practice, this map serves as a valuable reference for further understanding the recovery benefits of equine AM in corneal reconstruction.