1. Introduction References Methods Results – Normal CorneaResults - KeratoconusResults – Corneal Edema Results Keratoconus is a non-inflammatory, bilateral, progressive, often asymmetric, primary ectasia, associated with irregular astigmatism and decreased visual acuity. It results in progressive dissolution of Bowman’s membrane, the layer lying between the corneal epithelium and stroma. Cellular and structural changes in the cornea affect the integrity and lead to protrusion and scarring. Reported biochemical abnormalities in corneas of patients with keratoconus have included decreased protease inhibitors, increased keratocyte catalase activity, decreased alcohol dehydrogenase, decreased matrix metalloproteinase 8 (MMP- 8, collagenase), increased MMP-1, increased reactive oxygen/nitrogen species, higher hydrogen peroxide levels, and increased cathepsin K activity. Other abnormalities include absence of nerve growth factor receptor and absence of aquaporin 5. Since proteins are key players which execute nearly all downstream processes, proteomic analysis provides functional assessment of cellular processes. A recent report characterized the proteome of the normal human cornea and identified 141 distinct proteins; 86 of them were recognized in cDNA libraries from the corneas of patients with keratoconus. Furthermore, the bioinformatic analyses indicated the import of plasma proteins into the cornea. It is hypothesized that dysregulation of proteins in the corneas of patients with Keratoconus causes altered proteomic profile when compared to normal corneas. Normal and Keratoconus corneas Under an IRB approved protocol surgically discarded and de-identified normal donor and keratoconic corneas were obtained. The endothelium-Descemet’s membrane was removed from the cornea. The endothelium-free cornea was soaked in prewarmed 20mM EDTA in PBS solution for 30 minutes at 37º C. Forceps were used to separate the epithelial layer from the stroma. The samples were kept frozen until they are homogenized. The samples were homogenized using celLysis buffer (Sigma) and ultrasonicated for 30 seconds on ice. The homogenates were centrifuged at 10,000 rpm for 10 minutes and the supernatant was aliquoted and frozen at - 70ºC. Protein quantification of the samples was done by modified Lowry’s technique. The protein content of the samples was adjusted to 1mg/ml concentration. Corneal homogenates in the amount of 40µg were used to perform proteomic profiling by using Gold (Au) ProteinChips. Profiles were analyzed for protein peaks according to molecular weight using Ciphergen ProteinChip software.. Conclusions Gold (Au) ProteinChip array using SELDI-TOF-MS is a valuable technique to perform proteomic profiling of normal and keratoconic epithelial and stromal layers. The altered proteomic profile shows absence of peaks in the 20-150 Kd range in the keratoconic epithelium. However, there are distinct peaks in Keratoconus at 10.8, 12.7, 13.1, 14.6, 15.8 kDa which are absent in normals. Further studies are warranted to validate these results. The Normal corneal stroma contain peaks at 9.9, 14.5 in the 20kDa which are absent in Keratoconus. 1. US National Eye Institute, Facts about the Cornea and Corneal Disease Keratoconus. (http://www.nei.nih.gov/health/cornealdisease/index.asp# h). Accessed Feb 12 2006.http://www.nei.nih.gov/health/cornealdisease/index.asp# h 2. Ashwin PT, McDonnell. Collagen cross-linkage: a comprehensive review and directions for future research. Br J Ophthalmol 94:965-970, 2010. 3.Karring H, Thogersen IB, Klintworth GK, et al. The human cornea proteome: Bioinformatic analyses indicate import of plasma proteins into the cornea. Molecular Vision 12:451-460, 2006. 4.Attilano SR et al. Accumulation of mitochondrial DNA damage in keratoconus corneas. Invest Ophthalmol Vis Sci 46:1256-63, 2005. Acknowledgements: The Richard A. Perritt Charitable Foundation The normal corneal epithelium displayed a distinct proteomic profile in the 20Kd. However, unique peaks were observed at 10.1, 10.7 and 11.6 Kd SELDI-TOF-MS & Protein Chip THE PROTEOMIC PROFILE OF NORMAL AND KERATOCONIC EPITHELIAL AND STROMAL LAYERS BY PROTEINCHIP® USING SELDI-TOF-MS Omer Iqbal 2, MD, Daniel Kahn 2, BS, Shawn Aranha 2, MD, Samir Vira 1, MD, George Fisher 3, MS4, Bruce Gaynes 1, OD, PharmD, Jawed Fareed 2, PhD, Charles Bouchard 1, MD Departments of Ophthalmology 1 and Pathology 2, Loyola University Chicago, Stritch School of Medicine 3, Maywood, IL Normal - Epithelium MW range: 5-20 kDa; Intensity: 30 MW range: 20-150 kDa; Intensity: 6 Keratoconus - Epithelium MW range: 5-20 kDa; Intensity: 30 MW range: 20-150 kDa; Intensity: 6 Corneal Edema - Epithelium MW range: 5-20 kDa; Intensity: 30 MW range: 20-150 kDa; Intensity: 6 Normal - Stroma MW range: 5-20 kDa; Intensity: 5 MW range: 20-150 kDa; Intensity: 5 Keratconus - Stroma MW range: 5-20 kDa; Intensity: 5 MW range: 20-150 kDa; Intensity: 5 Corneal Edema - Stroma MW range: 5-20 kDa; Intensity: 5 MW range: 20-150 kDa; Intensity: 5