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Thomas Dyrlund, Ebbe Toftgaard Poulsen, Carsten Scavenius, Camilla Lund Nikolajsen, Ida B. Thøgersen, Henrik Vorum, Jan Enghild; Human cornea proteome: Identification and quantitation of the proteins of the three main layers including epithelium, stroma and endothelium. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1020.
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Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease.
The three main layers including the epithelium, stroma and endothelium of healthy human corneas were isolated and the proteins were (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. The resulting peptides were separated by LC-MS/MS and analysed on a TripleTOF 5600 mass spectrometer. Proteins were identified in the Swiss-Prot database using the Mascot algorithm and quantified using Mascot Distiller. Data extraction and processing was done using MS Data Miner.
A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis revealed that many of the identified proteins were human plasma proteins involved in the complement system, coagulation and defence against pathogen infections.
The separation of human corneas into the three main layers combined with modern mass spectrometry provides new insight into the proteins present in the individual layers and the relative abundance in each layer. This provides a useful reference dataset when exploring basic molecular mechanisms involved in corneal diseases, many of which are restricted to a specific corneal layer.
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