June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Human cornea proteome: Identification and quantitation of the proteins of the three main layers including epithelium, stroma and endothelium
Author Affiliations & Notes
  • Thomas Dyrlund
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Ebbe Toftgaard Poulsen
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Carsten Scavenius
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Camilla Lund Nikolajsen
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Ida B. Thøgersen
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Henrik Vorum
    Department of Ophthalmology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark
  • Jan Enghild
    Department of Molecular Biology, University of Aarhus, Aarhus C., Denmark
  • Footnotes
    Commercial Relationships Thomas Dyrlund, None; Ebbe Toftgaard Poulsen, None; Carsten Scavenius, None; Camilla Lund Nikolajsen, None; Ida B. Thøgersen, None; Henrik Vorum, None; Jan Enghild, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1020. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 481 cornea: endothelium • 482 cornea: epithelium • 484 cornea: stroma and keratocytes  
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