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H. Karring, I.B. Thogersen, G.K. Klintworth, J.J. Enghild, T. Moller–Pedersen; Proteome analysis of serum–cultured human corneal fibroblasts . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3831.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the most abundant water–soluble and insoluble proteins in human corneal fibroblasts to obtain a better understanding of the biochemical mechanisms involved in corneal wound healing and cellular transparency. Methods: Human corneal fibroblasts were obtained by explant technique and grown to third passage in medium containing 10% FCS. 2D gel electrophoresis was performed on the soluble fraction using various pH gradients. After silver staining, gel spots were analysed using MALDI mass spectrometry. The insoluble fraction was re–suspended in 70% TFA and digested with CNBr and trypsin to obtain peptide fragments suitable for ESI–MS and tandem MS analysis using Multidimensional protein identification technology (MudPIT). Results: About 300 of the most intense gel spots were identified from the soluble fraction, representing about 130 distinct proteins. The soluble fraction is dominated by proteins involved in processes such as metabolism, protein folding and degradation, cytoskeleton organisation and cell motility, secretion of proteins, cell division, and protection against oxidative stress. A significant number of the gel spots were identified as alpha–enolase, which previously has been classified as a lens enzyme–crystallin. No other major crystallin proteins were identified. Analysis of the insoluble fraction revealed several proteins from the cell– and organelle membranes. Conclusions: The protein expression profile shows that protein folding and degradation, cytoskeleton organisation, secretion of proteins, and protection against oxidative stress are dominating processes in cultured human corneal fibroblasts. Besides alpha–enolase, no major crystallins proteins are expressed in this keratocyte phenotype.
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