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X. Feng, R. Chaerkady, K. Kandasamy, C. Speck, A. Pandey, A. Jun, S. Chakravarti; Proteomic Profiling of Corneal Stroma in Keratoconus Patients. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3477.
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To quantify the different proteomic profiles of keratoconus patient and normal corneal stroma samples.
Corneas from normal individuals or patients with keratoconus were pooled and the epithelium from one half of each cornea was scraped off and stromal proteins extracted for mass spectrometry. The samples were trypsin-digested and the peptides labeled for quantitative proteomics with iTRAQ multiplex reagents (Applied Biosystems) followed by fractionation by strong cation exchange chromatography. Peptides from 24 fractions were then loaded onto a reversed phase capillary column and MS analyses were carried out on a high resolution Fourier transform mass spectrometer (LTQ-Orbitrap XL ETD, Thermo Scientific) in Pulsed Q Collision Induced Dissociation (PQD) mode. The iTRAQ reporter ion intensities were used for relative quantitation to identify proteins that were differentially expressed in keratoconus.
About 400 proteins were identified and quantified in the corneal samples, and their relative amounts estimated in keratoconus versus normal. About 121 proteins were over represented in keratoconus with ratios greater than 1.5; whilst 63 proteins were down regulated with ratios <=0.67. Proteins notably elevated in keratoconus include peroxiredoxin 5, transgelin 2, pro-alpha-1 collagen type 1, type V preprocollagen alpha 2 chain, fibronectin, apolipoprotein D precursor, prostaglandin H2 D-isomerase, thrombospondin 4 and fibrillin 1. Proteins that were significantly reduced in expression include collagen type XII, several crystallins (alpha B, beta A4 and gamma S), and fibromodulin. Interestingly, Collagen XII was reported by others to be decreased in keratoconus as well.
A majority of the differentially regulated proteins in keratoconus are known corneal components. Increased levels of collagen types I and V in keratoconus suggest architectural changes in the stromal ECM, while increased levels of fibronectin, thrombospondin 4, fibrillin 1 and fibulin 1 suggest altered ECM-stromal cell interactions. Down regulation of several crystalins also indicates keratocyte dysfunction in keratoconus.
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