Quantitative proteomics of aqueous and vitreous fluid from patients with idiopathic epiretinal membranes

Abstract

The purpose of the study was to perform a detailed, quantitative protein analysis of the aqueous (AF) and vitreous fluids (VF) from human eyes with idiopathic epiretinal membranes (iERM). The complementary approaches of quantitative liquid chromatography mass spectrometry (LCMS) and multiplex protein assays were utilised to reveal the protein composition in ocular fluids of this retinal disorder. In a prospective clinical trial, AF and VF was collected during surgery from twenty four eyes corresponding to twenty four patients with iERM. VF and AF from eight patients were labelled with the 4-plex iTRAQ reagent and analysed by LCMS. Each iTRAQ 4-plex experiment consisted of the AH and VH from two patients. A total of 323 proteins were identified in the AF and VF from eyes with iERM. Grouping the proteins according to involvement in biological processes, showed that the majority were involved in the classical and alternative pathway of complement activation (n = 27), proteolysis (n = 26) and cell adhesion (n = 28). iTRAQ relative quantitation revealed minimal variation in the protein content between both ocular compartments with only 3.96% of the identified proteins significantly, differentially-expressed. Eight proteins were expressed at a higher level in the VF compared to the AF; and 4 proteins were expressed at a lower level in the VF compared to the AF. For the multiplex bead assays, 29 growth factors and cytokines were assessed in the AF and VF from 16 additional patients with iERM. The protein profile was shown to be similar between VF and AF for the majority of factors except PDGF-AA. This factor was expressed at a higher level in the VF compared to the AF. The data presented in this study revealed that the majority of the proteins identified by LCMS and multiplex bead assays were present in both ocular compartments in similar quantities. This study is a first step, ‘discovery phase’ towards revealing and quantitating the protein content in the aqueous and vitreous fluid in human eyes with iERM.