Targeted Workflow Investigating Variations in the Tear Proteome by Liquid Chromatography Tandem Mass Spectrometry

Proteins in tears have an important role in eye health
and have
been shown as a promising source of disease biomarkers. The goal of
this study was to develop a robust, sensitive, and targeted method
for profiling tear proteins to examine the variability within a group
of healthy volunteers over three days. Inter-individual and inter-day
variabilities were examined to contribute to understanding the normal
variations in the tear proteome, as well as to establish which proteins
may be better candidates as eventual biomarkers of specific diseases.
Tear samples collected on Schirmer strips were subjected to bottom-up
proteomics, and resulting peptides were analyzed using an optimized
targeted method measuring 226 proteins by liquid chromatography-scheduled
multiple reaction monitoring. This method was developed using an in-house
database of identified proteins from tears compiled from high-resolution
data-dependent liquid chromatography tandem mass spectrometry data.
The measurement of unique peptide signals can help better understand
the dynamics of each of these proteins in tears. Some interesting
trends were seen in specific pathways or protein classes, including
higher variabilities for those involved in glycolysis, glutathione
metabolism, and cytoskeleton proteins and lower variation for those
involving the degradation of the extracellular matrix. The overall
aim of this study was to contribute to the field of tear proteomics
with the development of a novel and targeted method that is highly
amenable to the clinical laboratory using high flow LC and commonly
used triple quadrupole mass spectrometry while ensuring that protein
quantitation was reported based on unique peptides for each protein
and robust peak areas with data normalization. These results report
on variabilities on over 200 proteins that are robustly detected in
tear samples from healthy volunteers with a simple sample preparation
procedure.

泪液中的蛋白质在眼健康中扮演着至关重要的角色，并已被证实为疾病生物标志物的潜在来源。本研究旨在开发一种稳健、灵敏且具有针对性的方法，以对泪液蛋白质进行表征，从而考察一组健康志愿者在三天内的蛋白质变异性。研究分析了个体间和日间的变异性，旨在深入理解泪液蛋白质组中的正常变异，并确立哪些蛋白质可能成为特定疾病的最终生物标志物的更好候选者。在Schirmer滤纸上收集的泪液样本经过自下而上的蛋白质组学分析，所得肽段通过优化的靶向方法，通过液相色谱-预定多反应监测技术测量了226种蛋白质。该方法利用了公司内部数据库中从泪液的高分辨率数据依赖性液相色谱串联质谱数据中鉴定出的蛋白质。独特肽段信号的测量有助于更好地理解这些蛋白质在泪液中的动态变化。在特定的通路或蛋白质类别中观察到一些有趣的趋势，包括参与糖酵解、谷胱甘肽代谢和细胞骨架蛋白的蛋白质具有较高的变异性，而参与细胞外基质降解的蛋白质则变异性较低。本研究的总体目标是，通过开发一种新颖且具有针对性的方法，该方法在临床实验室中高度适用于使用高流量液相色谱和常用的三重四极杆质谱，同时确保蛋白质定量是基于每种蛋白质的独特肽段以及稳健的峰面积和数据归一化来报告的，为泪液蛋白质组学领域做出贡献。这些结果报告了在健康志愿者的泪液样本中，通过简单的样本制备程序稳健检测到的200多种蛋白质的变异性。