Human Cornea Proteome: Identification and Quantitation of the Proteins of the Three Main Layers Including Epithelium, Stroma, and Endothelium

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. In this
study, the three main layers including, the epithelium, stroma and
endothelium of healthy human corneas were isolated. Prior to analysis
by LC–MS/MS the proteins from the different layers were either
(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. 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 showed that many of the identified proteins are plasma proteins
involved in defense responses.