Keratoconus, an inherited corneal dystrophy which is characterized by progressive stromal thinning, severe nearsightedness, irregular astigmatism and scarring in the visual axes, is a leading cause of corneal transplantation in the US. Despite years of investigation into the genetics and biochemistry of keratoconus, we are only beginning to understand the primary causes and secondary pathobiology of this complex disease. Current investigations have targeted specific groups of molecules, and several labs have recently initiated studies to examine disease-specific changes in gene expression at the RNA level. However RNA and protein expression levels rarely correlate because of differences in synthesis and degradation rates, especially in a transcriptionally static tissue such as cornea. The ultimate function of the gene resides in the protein, and this function can be modulated by post-translational modifications such as alternative glycosylation and phosphorylation. Recent advances in 2D gel electrophoresis, mass spectrometry and proteomic bioinformatics have made it feasible for small academics labs to utilize proteomic technology. In addition to comparing relative expression levels of proteins in a mixed population, 2D gel electrophoresis also provides insight into the post-translational modifications of these proteins. The specific goal of this pilot and feasibility RO3 grant application is to use modern proteomic techniques to elucidate novel proteins whose expression is altered relative to controls in keratoconus. These proteins will be Identified using mass spectrometry, amino acid sequencing and proteomic database comparisons. Proteins which are not represented in public sequence databases will be further characterized by cDNA cloning. Identified proteins will be grouped into expression profiles according to possible roles in the pathobiology of this disease. It is expected that this pilot project will lead to the acquisition of a body of data which will significantly enhance our understanding of the pathobiology of corneal disease. This body of data will be made accessible to the community using a web-based interactive 2D database of corneal proteins.
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