QUANTITATIVE PROTEOMIC ANALYSIS OF AQUEOUS HUMOR IN PRIMARY OPEN ANGLE GLAUCOMA

Abstract

Purpose: To better understand the molecular mechanisms underlying primary open angle glaucoma (POAG) and to discover POAG biomarkers, we quantified proteomic change in aqueous humor (AH) from human POAG and control donors.

Methods: AH and plasma specimens were obtained from control patients undergoing cataract surgery and from POAG patients undergoing cataract or glaucoma surgeries at the Cole Eye Institute. Early experimental glaucoma (EEG) monkey plasma was obtained from Devers Eye Institute. AH was immunodepleted of abundant plasma proteins, reduced, alkylated, digested with trypsin and quantified by amino acid analysis. Peptides were labeled with iTRAQ tags, mixed, fractionated by strong cation exchange chromatography, and analyzed by LC MS/MS. Qualitative proteomic analysis of plasma utilized similar methods. Proteins were identified using the UniProtKB/Swiss-Protein human database; iTRAQ tags were quantified by the weighted averaged method using Mascot 2.4.1. Proteins were considered significantly altered if average ratios (POAG/ control) determined with ≥ 2 unique peptides in ≥ 3 specimens were at least 1 standard deviation from the mean with p values ≤ 0.05.

Results: Proteins (n = 425) were quantified in 4 POAG AH specimens relative to a pooled AH control from 6 normal donors. Among 18 significantly increased proteins in POAG AH were carbonic anhydrase 1, superoxide dismutase [Cu-Zn], and insulin-like growth factor-binding protein 7. Among 55 significantly decreased proteins were C-Jun-amino-terminal kinase-interacting protein 4, glutathione synthetase, synaptotagmin 5 and 10 different crystallins. Many of the significantly altered proteins in POAG AH were also detected in monkey EEG plasma (33%), in human POAG plasma (44%), and in the human plasma atlas (63%), supporting the possibility of monitoring these proteins for blood-borne biomarker validation.

Conclusions: Altered proteins in POAG AH may contribute to abnormalities in signal transduction, cellular defense mechanisms, and transport processes. Overall, these data may provide insight into the molecular mechanisms underlying glaucomatous damage to ocular tissues and blood-borne biomarker candidates for primary open angle glaucoma.

Keywords: 427 aqueous • 663 proteomics • 568 intraocular pressure