Label-free LC–MS/MS quantitative analysis of aqueous humor from keratoconic and normal eyes

PURPOSE The etiology of keratoconus (KC) and the factors governing its progression are not well understood. It has been suggested that this disease might be caused by biochemical alterations in the cornea; changes in the expression profiles of human aqueous humor (hAH) proteins have been observed in some diseases. To gain a new insight into the molecular mechanisms of KC pathology, we examined the hAH proteomes of those in the advanced stages of this disease. We used a high-throughput mass spectrometry approach to compare hAH protein expression in patients with KC and in control subjects. METHODS Aqueous humor samples were acquired from five keratoconus patients during keratoplasty surgery and from five myopic control subjects during phakic intraocular lens implantation. Quantitative mass spectrometry analysis using spectral counting was performed to determine the relative amounts of hAH proteins in the samples from KC patients and control individuals. RESULTS All KC patients included in the study presented severe keratoconus (K2 >52 D), and slit-lamp examination revealed microfolds in Descemet's membrane, without clinical signs of hydrops. We found significant differences between the expression levels of 16 proteins in the two groups. In KC samples, seven proteins were overexpressed and nine were underexpressed in comparison with the control group. Gene ontology analysis revealed that these deregulated proteins are implicated in several biologic processes, such as the regulation of proteolysis, responses to hypoxia, and responses to hydrogen peroxide, among others. CONCLUSIONS The protein expression profiles in hAH from KC patients and myopic control subjects differ significantly. This result suggests that some components of the hAH proteome are involved in this disease. Further in-depth analysis of the hAH proteome should provide a better understanding of the mechanisms governing the pathophysiology of KC.