Casey Eye Institute, Oregon Health & Science University, Portland, OR, USA.
Department of Chemical Physiology & Biochemistry, Oregon Health & Science University, Portland, OR, USA.
Ocul Surf. 2023 Apr;28:58-78. doi: 10.1016/j.jtos.2023.02.005. Epub 2023 Feb 9.
Human tears contain abundant, diverse sets of proteins that may serve as biomarkers of ocular surface health. There is a need for reproducible methods that consider multiple factors influencing the tear proteome, in addition to the variable of interest. Here we examined a workflow for proteomic analysis of tear proteins without the need to pool tear samples from multiple individuals, thus allowing for analyses based on individual factors, and increasing opportunities for protein biomarker discovery.
Tears were collected by Schirmer strip following topical ocular anesthetic application then individually stored at -80 °C prior to processing for proteomics. Tear proteins were extracted from Schirmer strips, digested using suspension trapping spin columns (S-Trap), and labeled with high multiplicity tandem mass tags (TMT). Peptide digests were then extensively fractionated by two-dimensional chromatography and analyzed by mass spectrometry to identify and measure changes in protein abundance in each sample. Analysis of select samples was performed to test protocols and to compare the impact of clinically relevant parameters. To facilitate comparison of separate TMT experiments, common pool samples were included in each TMT instrument run and internal reference scaling (IRS) was performed.
Differences in subsets of tear proteins were noted for: geographic site of tear collection, contact lens use, and differences in tear fluid volume among individuals.
These findings demonstrate that proteomic analysis of human tear proteins can be performed without the need to pool samples, and that development of analytic workflows must consider factors that may affect outcomes in studies focused on diverse clinical samples.
人泪中含有丰富多样的蛋白质组，可作为眼表健康的生物标志物。需要有可重复的方法，除了感兴趣的变量外，还要考虑影响泪液蛋白质组的多个因素。在这里，我们研究了一种无需从多个个体中汇集泪样即可进行蛋白质组学分析的工作流程，从而允许基于个体因素进行分析，并增加蛋白质生物标志物发现的机会。
在局部眼部麻醉后，用 Schirmer 条收集眼泪，然后单独储存在-80°C 下，再进行蛋白质组学处理。从 Schirmer 条中提取泪液蛋白，使用悬浮捕获自旋柱（S-Trap）进行消化，并使用高多重串联质量标签（TMT）进行标记。然后通过二维色谱法对肽消化物进行广泛分级，并通过质谱分析来鉴定和测量每个样品中蛋白质丰度的变化。对选定的样本进行分析以测试方案并比较临床相关参数的影响。为了便于比较单独的 TMT 实验，在每个 TMT 仪器运行中都包含公共池样本，并进行内部参考标度（IRS）。
在以下方面观察到泪液蛋白的亚群存在差异：泪液采集的地理位置、隐形眼镜的使用以及个体间泪液量的差异。
这些发现表明，无需汇集样本即可进行人类泪液蛋白质的蛋白质组学分析，并且分析工作流程的开发必须考虑可能影响聚焦于不同临床样本的研究结果的因素。
