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F. E. Baltasar, E. H. Leung, O. Iqbal, J. Fareed, E. Coyne, P. Bu, C. S. Bouchard; Proteomic Analysis of the Keraotoconus Cornea by ProteinChip® Array. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4547.
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The etiology of keratoconus remains unknown and may be linked to dysfunction and dysregulation of the stromal and/or epithelial layers of the cornea. The proteomic profile of the cornea has been previously described by methods that include peptide mass fingerprinting, Liquid Chromatography Tandem Mass Spectroscopy (LC-MS/MS), 2D Polyacrylamide gel electrophoresis (PAGE), 1D PAGE, strong cation exchange (SCX) of peptides, and MALDI TOF MS. This study aims to display differences in the proteomic profile of normal and keratoconus corneas by ProteinChip® array (BioRad). ProteinChip® Array employs Surface Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry (SELDI-TOF MS).
Normal human corneas were obtained from the Illinois Eye Bank, and abnormal corneas were obtained from consenting donors undergoing corneal transplant following an IRB-approved protocol. Corneas were homogenized in 50mM HEPES, 100mM NaCl, 2mM DTT, 1mM Sodium Ortho vanadate, 10% Triton X, and 200µL Protease Inhibitor Concentrate. Corneal homogenates were first analyzed by SDS-PAGE. The proteomic profiles of 40µg of corneal homogenate were then obtained by SELDI TOF MS using strong anion exchange (Q10) and Gold (Au) ProteinChips. Profiles were analyzed for protein peaks according to molecular weight using Ciphergen ProteinChip software.
Normal corneas display a distinct proteomic profile in the <25kDa weight range. Mass spectrometry with the Q10 chip shows prominent peaks at 10.0kDa and 16.6kDa respectively in normal corneas. In keratoconus, two unique high intensity peaks are noted in the 11-12kDa and 13-14kDa weight range which are not present in normal corneas.
ProteinChip array is a useful method for the analysis of corneal proteins and allows for the successful comparison of normal and diseased corneal proteomes. The presence of unique peaks in the proteomic profile of the keratoconus cornea suggests a pathologic dysregulation of corneal proteins.
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