Quantitative protein-antibody array analysis of aqueous humor In glaucoma and cataract

Abstract

Purpose: To understand the underlying cause of glaucoma by comparing levels of aqueous humor proteins of subjects with glaucoma versus those with cataract. In a previous study, we identified 8 proteins that were significantly elevated in glaucoma aqueous. The goal of this study was to validate our previous findings with a separate sample set and to identify additional candidate proteins that differentiate glaucoma and cataract aqueous humor using protein-antibody arrays.

Methods: Aqueous samples were obtained from eyes during glaucoma or cataract surgery. Fifty glaucoma and 50 cataract samples were age- and gender-matched. A third aqueous sample group (N=18) consisted of cataract patients without glaucoma who received latanoprost or timolol eyedrops for two weeks prior to surgery. Aqueous samples were incubated on customized glass slides (RayBiotech Quantibody Arrays) spotted with antibodies to 40 proteins previously identified in aqueous humor and including the 8 proteins we previously found to be increased in glaucoma. Signals were visualized with a fluorescence laser scanner. A mixed effects model fit with restricted maximum likelihood estimation was used to test the differential abundance of each protein. Multiple comparison correction was performed using the Benjamini-Hochberg method.

Results: The glaucoma and cataract groups were similar in age (70.8 vs. 67.1, p=0.07), gender (24 vs. 30 males, p=0.32), and race (11 vs.7 blacks, p=0.44). After multiple comparison correction, at least 14 proteins were found to be increased in glaucoma, including all 8 previously identified proteins (insulin-like growth factor binding protein 3, angiopoietin 2, eotaxin, insulin-like growth factor binding protein 2, neural cell adhesion molecule 1, macrophate colony stimulating factor 1 receptor, matrix metalloproteinase 3, and interleukin 8). No differences were found between cataract groups with or without glaucoma medications.

Conclusions: On two large and separate sets of aqueous samples, we have identified and verified the statistically significant increased abundance of 8 proteins in glaucoma aqueous humor. We have also identified several other candidate proteins that distinguish glaucoma aqueous from cataract aqueous. Subtle differences detected in aqueous humor protein profiles between glaucoma and cataract may help elucidate the pathophysiology of glaucoma.

Keywords: 663 proteomics • 427 aqueous • 445 cataract