Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University , Kowloon, Hong Kong, China.
SCIEX, Hong Kong, China.
J Proteome Res. 2017 Oct 6;16(10):3753-3765. doi: 10.1021/acs.jproteome.7b00449. Epub 2017 Sep 27.
The molecular pathophysiology of corticosteroid-induced ocular hypertension (CIH) is not well understood. To determine the biological mechanisms of CIH, this study investigated protein expression profiles of human trabecular meshwork (hTM) cells in response to dexamethasone and prednisolone treatment. Both discovery-based sequential windowed data independent acquisition of the total high-resolution mass spectra (SWATH-MS) and targeted based high resolution multiple reaction monitoring (MRM-HR) confirmation were applied using a hybrid quadrupole-time-of-flight mass spectrometer. A comprehensive list of 1759 proteins (1% FDR) was generated from the hTM. Quantitative proteomics revealed 20 differentially expressed proteins (p-value ≤ 0.05 and fold-change ≥ 1.5 or ≤ 0.67) commonly induced by prednisolone and dexamethasone, both at 300 nM. These included connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1), two proteins previously implicated in ocular hypertension, glaucoma, and the transforming growth factor-β pathway. Their gene expressions in response to corticosteroids were further confirmed using reverse-transcription polymerase chain reaction. Together with other novel proteins identified in the data sets, additional pathways implicated by these regulated proteins were the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, integrin cell surface interaction, extracellular matrix (ECM) proteoglycans, and ECM-receptor interaction. Our results indicated that an integrated platform of SWATH-MS and MRM-HR allows high throughput identification and confirmation of novel and known corticosteroid-regulated proteins in trabecular meshwork cells, demonstrating the power of this technique in extending the current understanding of the pathogenesis of CIH.
皮质类固醇诱导性眼压升高（CIH）的分子病理生理学尚不清楚。为了确定 CIH 的生物学机制，本研究调查了人眼小梁细胞（hTM）对地塞米松和泼尼松龙治疗的蛋白质表达谱。使用混合四极杆飞行时间质谱仪，应用基于发现的顺序窗口数据独立采集的总高分辨率质谱（SWATH-MS）和基于靶向的高分辨率多重反应监测（MRM-HR）确证。从 hTM 生成了 1759 种蛋白质的综合清单（1% FDR）。定量蛋白质组学揭示了 20 种差异表达的蛋白质（p 值≤0.05，倍数变化≥1.5 或≤0.67），这些蛋白质通常由 300 nM 的泼尼松龙和地塞米松共同诱导。其中包括结缔组织生长因子（CTGF）和血小板反应蛋白-1（THBS1），这两种蛋白以前都与眼压升高、青光眼和转化生长因子-β途径有关。使用逆转录聚合酶链反应进一步证实了它们对皮质类固醇的基因表达。与数据集中鉴定的其他新蛋白质一起，这些受调控蛋白质所涉及的其他途径包括磷脂酰肌醇 3-激酶（PI3K）-蛋白激酶 B（Akt）信号通路、整合素细胞表面相互作用、细胞外基质（ECM）蛋白聚糖和 ECM-受体相互作用。我们的结果表明，SWATH-MS 和 MRM-HR 的集成平台允许高通量鉴定和确认小梁细胞中新型和已知的皮质类固醇调节蛋白，证明了该技术在扩展 CIH 发病机制的当前理解方面的强大功能。
