Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium

Abstract

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.

Figure 1

Venn diagram showing the number of overlapping proteins identified in the three layers of the human cornea. A total of 3250 unique proteins were identified, 2737 in the epithelial, 1679 in the stromal and 880 in the endothelial layer.

Figure 2

Venn diagram showing the number of overlapping proteins identified in human cornea by (i) SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digest without prior protein separation or (iii) in-solution digest followed by cation exchange chromatography. A total of 2958 unique proteins were identified by SDS-PAGE separation, 1010 by in-solution digest without prior protein separation and 1729 by in-solution digest followed by cation exchange chromatography.

Figure 3

Venn diagram showing the number of overlapping proteins quantified in the three layers of the human cornea. Numbers in brackets are quantified proteins based on gel separation of proteins and the other number is for the in-solution digests. A total of 771 proteins were quantified, 157 based on in-solution digest and 770 based on gel separations. Total proteins quantified for the individual layers are epithelium, 110 [663]; stroma, 44 [342] and endothelium, 59 [140].

Figure 4

Gene Ontology distributions for summary categories based on cellular component of stroma proteins. The IDs bars represent the percentage of identified proteins in the summary category and the amount bars represent the percentage of quantified proteins accounting for the relative amount of each protein. Distributions for all layers and ontologies are provided in Supporting Information 3.

Figure 5

Quantified proteins for the three different cornea layers were classified into 3 broad cellular component categories. The bars represent the percentage of quantified proteins accounting for the relative amount of each protein.

Figure 6

Venn diagram showing the number of overlapping proteins identified in human cornea, tear and aqueous humor. A total of 990 unique Swiss-Prot proteins have previously been identified in human tear and 361 in human aqueous humor.^,