Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China.
Centre for Eye and Vision Research (CEVR), 17W Hong Kong Science Park, Hong Kong, China.
Int J Mol Sci. 2021 Apr 29;22(9):4721. doi: 10.3390/ijms22094721.
Most of the previous myopic animal studies employed a single-candidate approach and lower resolution proteomics approaches that were difficult to detect minor changes, and generated limited systems-wide biological information. Hence, a complete picture of molecular events in the retina involving myopic development is lacking. Here, to investigate comprehensive retinal protein alternations and underlying molecular events in the early myopic stage, we performed a data-independent Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) based proteomic analysis coupled with different bioinformatics tools in pigmented guinea pigs after 4-day lens-induced myopia (LIM). Myopic eyes compared to untreated contralateral control eyes caused significant changes in refractive error and choroid thickness ( < 0.05, = 5). Relative elongation of axial length and the vitreous chamber depth were also observed. Using pooled samples from all individuals ( = 10) to build a species-specific retinal ion library for SWATH analysis, 3202 non-redundant proteins (with 24,616 peptides) were identified at 1% global FDR. For quantitative analysis, the 10 individual retinal samples (5 pairs) were analyzed using a high resolution Triple-TOF 6600 mass spectrometry (MS) with technical replicates. In total, 37 up-regulated and 21 down-regulated proteins were found significantly changed after LIM treatment (log2 ratio (T/C) > 0.26 or < -0.26; ≤ 0.05). Data are accepted via ProteomeXchange with identifier PXD025003. Through Ingenuity Pathways Analysis (IPA), "lipid metabolism" was found as the top function associated with the differentially expressed proteins. Based on the protein abundance and peptide sequences, expression patterns of two regulated proteins ( and ) identified in this pathway were further successfully validated with high confidence ( < 0.05) using a novel Multiple Reaction Monitoring (MRM) assay on a QTRAP 6500+ MS. In summary, through an integrated discovery and targeted proteomic approach, this study serves as the first report to detect and confirm novel retinal protein changes and significant biological functions in the early LIM mammalian guinea pigs. The study provides new workflow and insights for further research to myopia control.
大多数先前的近视动物研究采用了单一候选方法和较低分辨率的蛋白质组学方法，这些方法难以检测到微小的变化，并且只能产生有限的系统范围的生物学信息。因此，涉及近视发展的视网膜分子事件的全貌尚不清楚。在这里，为了研究早期近视阶段中视网膜的全面蛋白质变化和潜在的分子事件，我们在 4 天镜片诱导性近视（LIM）后对色素豚鼠进行了基于无依赖数据的顺序窗口采集所有理论质谱（SWATH）的蛋白质组学分析，并结合了不同的生物信息学工具。与未经处理的对侧对照眼相比，近视眼引起了明显的屈光不正和脉络膜厚度变化（<0.05，=5）。还观察到轴向长度的相对伸长和玻璃体腔深度。使用来自所有人（=10）的混合样本构建用于 SWATH 分析的物种特异性视网膜离子文库，在 1%的全局 FDR 下鉴定出 3202 种非冗余蛋白质（带有 24616 种肽）。对于定量分析，使用高分辨率 Triple-TOF 6600 质谱仪（MS）对 10 个个体视网膜样本（5 对）进行了技术重复分析。总共发现 LIM 处理后有 37 种上调和 21 种下调蛋白发生明显变化（对数比（T/C）>0.26 或<-0.26；≤0.05）。数据通过 ProteomeXchange 以标识符 PXD025003 接受。通过 IPA 分析，发现“脂质代谢”是与差异表达蛋白相关的最重要功能。基于蛋白质丰度和肽序列，该途径中鉴定的两种调节蛋白（和）的表达模式使用 QTRAP 6500+ MS 上的新型多重反应监测（MRM）测定法进一步成功地以高置信度（<0.05）进行了验证。总之，通过综合的发现和靶向蛋白质组学方法，本研究首次报道了在早期 LIM 哺乳动物豚鼠中检测和确认新型视网膜蛋白质变化和重要生物学功能。该研究为近视控制的进一步研究提供了新的工作流程和见解。
