Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hong Kong.
Centre for Eye and Vision Research (CEVR), 17W, Hong Kong Science Park, Hong Kong.
Int J Mol Sci. 2024 Oct 3;25(19):10644. doi: 10.3390/ijms251910644.
The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 ( = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes ( = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed ( < 0.05, = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period.
玻璃体（VH）是一种透明的凝胶状物质，占据眼球的三分之二，并在眼球延长过程中发生最大变化。对 VH 正常生长期间的定量蛋白质组学研究可以为理解近视早期其进展机制提供新的见解。在这项研究中，结合数据非依赖性采集（SWATH-MS）和靶向 LC-ESI-MS/MS 来鉴定和定量鸡模型中 VH 在正常生长期间（4、7、14、21 和 28 天龄）的相对蛋白变化。小鸡在正常生长条件（12/12 h 明暗循环）下饲养 28 天，在第 4 天（基线）、第 7、14、21 和 28 天进行眼部测量，包括屈光和生物测量（=每个时间点 6 只小鸡）。从单个动物中提取的玻璃体蛋白进行消化，并在每个时间点将其分为左眼池和右眼池进行蛋白分析。使用信息依赖性采集（IDA）方法通过组合来自各个时间点的注射，生成鸡的玻璃体蛋白质组。使用单个池样本，通过 SWATH-MS 对每个时间点之间的蛋白质进行定量。随后，在第 7 天和第 14 天之间使用 MRMHR 单独对随机眼（=4 只）的单独批次动物中确认 DEPs。在这段时间内，屈光度和玻璃体腔深度（VCD）发现明显变化（<0.05，每个时间点 6 只）。建立了一个全面的玻璃体蛋白离子文库，其中包含在 1%假发现率（FDR）下鉴定的 1576 个非冗余蛋白（22987 个独特肽）。与第 7 天相比，使用 SWATH-MS 在所有时间点都发现了 12 个上调和 26 个下调的蛋白。在这种实验条件下，使用 MRMHR 首次验证了几个 DEPs，如甲胎蛋白、钙黏蛋白家族、神经粘蛋白和 reelin，它们参与了结构和生长相关途径。这项研究为小鸡在正常生长期间提供了玻璃体的第一个全面光谱文库，并使用 SWATH-MS 和 MRMHR 在正视化期间提供了一组潜在的生长相关蛋白生物标志物候选物。
