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L Fedorova, F Shang, HK Wu, A Ashrafzadeh, NV Laver, A Taylor, ME Fini, PF Olson; Down-Regulation of Two Proteins in Keratoconus Revealed by Comparative Proteomic Analysis of Human Corneal Tissue . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3238.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:In keratoconus the cornea assumes a conical shape due to non-inflammatory thinning. Severely-affected patients require transplantation because of corneal scarring. Despite years of investigation, we have little knowledge of the genetic and biochemical causes of keratoconus. We have used comparative proteomics to define changes in protein expression between normal (N) and keratoconic (KC) corneas. Methods:KC corneal buttons were obtained after penetrating keratoplasty. Normals were obtained from the New England Eye and Tissue Transplant Bank or upon autopsy. Whole corneal buttons were pulverized in lN2 and homogenized in a sequential series of extraction buffers. 200 µg of protein was focused on 17cm IEF strips (BioRad) for 90kV-Hrs and separated by SDS-PAGE. Comparative gels were stained with silver, and preparative gels were stained with SyproRuby. Prior to mass spec, protein spots were excised and digested with trypsin. Eluted peptides were analyzed by MS/MS on an ion trap mass spectrometer. Peptide mass fingerprinting was performed using the program PeptIdent (ExPASy). Results:Approx. 50% of corneal proteins extracted with buffer 1 (40 mM Tris base), 40% with buffer 2 (8 M Urea, 4% CHAPS, 40 mM Tris base, 0.2% Bio-Lyte 3/10) and 10 with buffer 3 (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB (3-10), 40 mM Tris, 0.2% Bio-Lyte 3-10). Approx.150 protein spots from extract 2 can be clearly distinguished in the area defined by pI 4-7 and <40 kD. Severe horizontal and vertical streaking is present above 40 kD, due to highly charged proteoglycans (PGs). Comparison of proteins extracted from 15 N and 2 KC corneas showed that: approx. 80% of proteins are expressed at consistent levels in normal corneas of diverse genetic origins and at least eight proteins are differentially-expressed between KC and N. 2 of these spots were present at very high levels in all 15 normals and either absent or barely visible in the 2 KC corneas analyzed thus far. Preliminary peptide fingerprinting data suggest that these two proteins are cytokines involved in mediating inflammatory response to infection. Conclusion:Proteomic analysis of the cornea presents unique challenges because of the presence of highly-charged PGs. We have worked out extraction and separation conditions that allow good resolution of small (<40kD) proteins. Proteomics has revealed differential protein expression between N and KC corneas, and we will expand our study to include more KC as well as other diseased corneas.
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