Proteome profiling and relative quantification of protein expressions in the chick vitreous and retina during compensated ocular growth

Abstract

The high prevalence of myopia and its related complications of myopia pose a heavy burden to society and individuals. To date, uncorrected refractive error is recognized as the major cause of avoidable visual impairment globally. Rapid development of mass spectrometry (MS) and quantitative proteomic methods have made proteomics a tool of choice for studying biomarkers and pathogenesis in many diseases. In this study, the advanced MS coupled to novel proteomic techniques were applied in myopia research. Identification of proteins in a tissue is the fundamental and essential step in typical proteomics. Using one-dimensional gel electrophoresis (1D gel) followed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry (nanoLC-ESI-ionTrap MS/MS), a total of 341 and 1021 distinct proteins were identified in normal growing chick vitreous and retina, respectively, by searching against the International Protein Index (IPI) chick protein database. Among them, 196 common proteins were found shared. The constituent of proteins in these two tissues were found similar when they were classified according to their known biological activities (BP) and molecular functions (MF). The major difference between these two tissues was in cellular components in which relatively more proteins in the vitreous were in extracellular region and extracellular matrix, while the majority of retinal proteins were in cell part and organelle. These protein profiles set as fundamental ground works for follow up studies. Differentially expressed proteins during ocular growth may provide new insights into the pathogenesis of myopia. Quantitative proteomics using Isotope Coded Protein Labeling (ICPL) technique was applied in studying the ametropic vitreous (+10D/-10D lens inducement for 3 and 7 days). Four proteins (apolipoprotein A1, purpurin, cystatin and ovotransferrin) were detected with up-regulated expression levels in the myopic eye after wearing -10D lenses for 3 days. It was the first report of up-regulated expression of Apolipoprotein A1 in lens-induced myopic chick vitreous. Network analysis using Ingenuity Pathway Analysis (IPA) software indicated that anoxidative stress and lipid metabolism may be involved in the myopic progression. Furthermore, transforming growth factor beta (TGF-{490}) signaling pathway may also participate in regulating ocular growth with its connection to Apolipoprotein A1 and cystatin. Quantitative proteomics was also performed in chick retina wearing -10D/+10D lenses (3 and 7 days) and diffuser lenses (7 hours, 3 and 7 days). Based on the differentially expressed proteins, it was found that the retinal protein targets were mainly of cytoplasmic origin in both paradigms. According to the bioinformatics analysis, various metabolisms and glycolysis process were suggested to regulate or participate in myopic ocular growth. Moreover, several common proteins found in both lens-inducement and form-deprivation suggested possible common signaling pathways shared between these two paradigms. On the other hand, ApoA1 was found down-regulated after lens inducement but not significantly changed after form deprivation, which could suggest a co-existence of independent pathways. In addition, protein pathway analysis of all differentially expressed retinal protein further revealed insulin signaling pathway that was previously reported to be related to myopia progression. In addition to studying protein abundance levels in the chick retina, the feasibility of applying phosphoproteomic technology in myopia research was explored in short term lens treatment (-10D/+10D, 3 days). By using a titanium dioxide enrichment approach coupled to a next generation nanoLC Triple Time of Flight MS, a total of 290 proteins (381 phosphopeptides) in myopic retina with 401 phosphorylation sites were detected. In the hyperopic retina, 249 proteins (347 phosphopeptides) with 372 phosphorylation sites were detected. Majority of detected phosphopeptides using TiO₂ enrichment was single-phosphorylated, with a small minority detected as double-phosphorylation (the ratio is approximately 9:1). Peptides with triple-phosphorylation were rare. This application provided a new and complementary research direction for future myopia study. Using both discovery profiling and targeted proteomics verification, the study supported some protein candidates which were reported by previous myopia studies. A number of novel protein targets were also found with possible regulatory pathways suggested by emerging bioinformatics tool. High-throughput quantitative proteomics was proven as a vital tool in searching for new therapeutic options for future myopia study.