Quantitative proteomics analysis of human trabecular meshwork (hTM) in response to corticosteroids using SWATH™-MS

Abstract

Purpose : Corticosteroids can increase the intraocular pressure (IOP) of the eye and prolonged treatment with steroids can even lead to glaucomatous optic neuropathy. To better understand the underlying biological mechanisms of corticosteroid-induced ocular hypertension (CIH), differential protein expression profiles of human trabecular meshwork (hTM) cells after treated with dexamethasone (DEX) and prednisolone (PRED) were investigated. Methods : Primary human TM cell cultures from 3 healthy donors were incubated with and without DEX or PRED for 7 days. Differentially expressed proteins were identified and quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS; with a high-resolution Quadrupole-time-of-flight (QTOF) instrument) using sequential windowed acquisition of all theoretical fragment ions (SWATHTM) technologies. Results : A total of 1759 non-reductant proteins were identified using Protein Pilot 5.0 analysis (at 1% FDR) that formed a hTM proteome library for quantitative SWATHTM data analysis. As compared to controls, twenty proteins were differentially regulated in both DEX- and PRED-treated hTM cells (P<0.05, fold change ≥ 1.5 folds and not less than 2 peptide matches per protein). The molecular functions of these proteins, as deduced by PANTHER gene list analysis, were related to translational regulation, binding, receptor, enzyme regulation, structural molecule, catalytic and transporter. In addition, 5 most important pathways were identified from iPathwayGuideTM analysis as: TGF-β signaling pathway, PI3K-Akt signaling pathway, focal adhesion, proteoglycans in cancer and ECM-receptor interaction. Furthermore, 8 out of 20 common candidates were affected by both corticosteroids and they showed pathway 'connection' by STRING software which may be important in the pathogenesis of glaucoma. Conclusions : The results demonstrate significant protein changes in hTM cells in response to corticosteroids and reveal similarities in proteomic changes induced by the two different corticosteroids. By using online bioinformatics approaches, this study demonstrates an exhaustive picture on protein changes after corticosteroids treatment in hTM cells. This new generation proteomic approach is able to comprehensively identify novel as well as known protein changes in CIH and helps extend the current understanding of pathogenesis of CIH.