Department of Ophthalmology, Medical University Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria.
Exp Eye Res. 2013 Mar;108:48-58. doi: 10.1016/j.exer.2012.11.010. Epub 2012 Nov 29.
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.
本研究旨在对特发性眼内细胞纤维膜（iERM）患者的房水（AF）和玻璃体液（VF）进行详细的、定量的蛋白质分析。本研究采用定量液相色谱-质谱联用（LCMS）和多重蛋白检测分析方法，以揭示这种视网膜疾病眼内液的蛋白质组成。在一项前瞻性临床试验中，从 24 例 iERM 患者的手术中收集了 AF 和 VF。8 例患者的 VF 和 AF 用 4 重 iTRAQ 试剂标记，并通过 LCMS 进行分析。每个 iTRAQ 4 重实验均由两位患者的 AH 和 VH 组成。在 iERM 眼中的 AF 和 VF 中鉴定出 323 种蛋白质。根据参与的生物学过程对蛋白质进行分组，结果表明，大多数蛋白质涉及经典和替代补体激活途径（n=27）、蛋白水解（n=26）和细胞黏附（n=28）。iTRAQ 相对定量显示，两个眼部分间的蛋白质含量变化很小，只有 3.96%的鉴定蛋白存在显著差异表达。8 种蛋白在 VF 中的表达水平高于 AF；4 种蛋白在 VF 中的表达水平低于 AF。对于多重 bead 检测，在另外 16 例 iERM 患者的 AF 和 VF 中评估了 29 种生长因子和细胞因子。结果表明，除 PDGF-AA 外，大多数因子在 VF 和 AF 之间的蛋白谱相似。该因子在 VF 中的表达水平高于 AF。本研究中提供的数据表明，通过 LCMS 和多重 bead 检测鉴定的大多数蛋白在两个眼部分中以相似的量存在。本研究是揭示和定量 iERM 患者眼内液中蛋白质含量的第一步，即“发现阶段”。
