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首页> 外文期刊>BMC Ophthalmology >Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration
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Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

机译：患有年龄相关性黄斑变性的玻璃疣和网状假疣患者房水定量蛋白质组学分析

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To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls. After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups). Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen. Differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.
机译：为了鉴定与干性黄斑变性（AMD）发病机理相关的新型生物标记，我们采用了人类视网膜色素上皮（RPE）细胞培养模型，该模型模仿了干性AMD的某些特征，包括RPE内和RPE沉积物的积累。然后，我们使用数据独立的采集方法（所有理论碎片离子质谱的顺序窗口采集）研究了干燥的AMD患者和对照的房水（AH）蛋白质组。建立人胎原代RPE（hfRPE）细胞的色素均匀着色的极化单层后，将细胞暴露于4-羟基-2-壬烯醛（4-HNE），然后进行蛋白质印迹，免疫荧光分析和细胞或条件培养基的ELISA，几种感兴趣的蛋白质。根据11名干燥的AMD患者的表型（包括软性玻璃疣和网状假性脾脏[RPD]）和2名对照（3组），进行了数据依赖的采集以识别AH蛋白质组和基于SWATH的质谱。与基于APOA1，组织蛋白酶D和簇蛋白免疫反应性的对照培养相比，在4-HNE处理的hfRPE细胞中观察到了RPE内和RPE沉积物的增加。另外，在有或没有进行4-HNE处理的情况下，顶端和基底腔中蛋白质的丰度差异证实了hfRPE细胞中蛋白质的极化分泌。通过SWATH-MS对干燥的AMD患者和对照中的119种蛋白质进行了定量。在这三组蛋白质中，有65种蛋白质的丰度发生了显着变化。生成了二维主成分分析图，以鉴定与干燥AMD发病机理相关的典型蛋白质。在鉴定出的蛋白质中，包括APOA1，CFHR2和CLUS在内的八种蛋白质以前被认为是玻璃疣的主要成分或调节剂。以前尚未将三种蛋白质（SERPINA4，LUM和KERA蛋白质）描述为玻璃疣的成分或与干性AMD相关。有趣的是，与细胞外基质组织有关的LUM和KERA蛋白在RPD和软性玻璃疣中均被上调。在本研究中，使用SWATH-MS量化了玻璃疣和RPD患者之间AH中差异蛋白的表达。对干燥的AMD患者进行详细的蛋白质组学分析可能会提供对玻璃疣和RPD的体内生物学的见解。

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