Proteomic Profiles Of Aqueous Humor In Glaucoma And Cataract

Abstract

Purpose: : To understand the underlying cause of glaucoma by comparing the aqueous humor proteins of subjects with glaucoma versus those with cataract.

Methods: : Aqueous samples were obtained from eyes during glaucoma or cataract surgery. Liquid chromatography tandem mass spectrometry (LC-MS/MS) provides great depth of protein coverage, while protein antibody arrays are used for lower abundance proteins. Glaucoma and cataract samples were age and gender matched. For LC-MS/MS, aqueous samples were depleted of albumin and immunoglobulins. Tryptic peptides were analyzed on a linear ion-trap (LTQ) mass spectrometer. After peptide identification, peptide-level quantitative information was combined into protein summaries based on reference sequence databases. For protein antibody arrays, aqueous samples were incubated on customized glass slides spotted with antibodies to 40-50 proteins previously identified in aqueous humor. 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: : From 21 cataract and 20 glaucoma samples LC-MS/MS detected 82 proteins with high confidence, all of which had been previously found in aqueous humor. For most proteins, mean intensity levels between glaucoma and cataract samples differed by less than 10%. After adjustment for multiple comparisons, alpha-2 macroglobulin was found to be significantly different between the glaucoma and cataract samples. Two sets of protein antibody array analysis were performed. In the first set (21 glaucoma and 21 cataract samples), although no proteins were significantly different after multiple comparison correction, at least seven proteins showed potential for further study. We included these seven proteins with an additional 33 proteins in our second set of protein antibody arrays using a larger sample size (50 cataract and 50 glaucoma samples). After adjusting for multiple comparisons and excluding one outlier, we identified 8 proteins, including interleukin-8, that were significantly higher in abundance in the glaucoma samples compared to the cataract samples.

Conclusions: : In these studies, over 150 proteins were compared between glaucoma and cataract aqueous humor samples. Nine proteins, including alpha-2 macroglobulin and interleukin-8 were found to be significantly different in abundance. Subtle differences detected in aqueous humor protein profiles between glaucoma and cataract may help elucidate the pathophysiology of glaucoma.

Keywords: proteomics • aqueous • outflow: trabecular meshwork