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Omer Iqbal, Daniel Kahn, Shawn Aranha, Bruce I. Gaynes, Jawed Fareed, Charles S. Bouchard; The Proteomic Profile of Normal and Keratoconic Epithelial and Stromal Layers by Proteinchip using Seldi-Tof-Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4185.
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Keratoconus is a non-inflammatory, bilateral, progressive, often asymmetric primary ectasia associated with irregular astigmatism and decreased visual acuity. It is one of the indications for corneal transplantation. The pathogenesis of this disease is not completely understood. In the present study the proteomic profiling of epithelial and stromal layers of normal and keratoconic corneas was performed using SELDI-TOF-MS.
Under an IRB approved protocol surgically discarded and de-identified normal donor (n=2) and keratoconic corneas (n=2) 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 samples were adjusted to 1mg concentration. Corneal homogenates in the amount of 40ug 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.
The normal corneal epithelium displayed a distinct proteomic profile in the <25Kd molecular weight range. Mass spectrometry with the Gold (Au) chip shows prominent peaks at 5.0, 7.0, 7.7, 8.5, 10.1, 11.6, 12.3, 15 and 17.9 Kd. The stromal layer showed prominent peaks at 8.5, 9.4, 9.8 and 11.0 Kd. However, the peaks in the 20-150 Kd range which were present in the normals were not observed in keratoconic epithelial layer. The epithelial layer from corneal edematous cornea showed similar peaks to that of normals at >20Kd. However, unique peaks were observed at 10.1, 10.7 and 11.6 Kd.
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 manifested by absence of peaks in the 20-150 Kd range in the keratoconic epithelium is crucial to the loss of structural integrity of cells resulting in Keratoconus.
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