Center for Biotechnology and Genomic Medicine, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
Department of Ophthalmology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA.
Int J Mol Sci. 2022 Feb 19;23(4):2307. doi: 10.3390/ijms23042307.
The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.
泪膜是覆盖眼睛角膜和结膜上皮的多层流体，提供润滑、营养，并保护眼睛免受外界环境的伤害。泪液中含有高浓度的蛋白质，因此由于其接近眼表面的病变部位以及采集的非侵入性性质，已被认为是眼部疾病生物标志物的潜在来源。对于干眼症等疾病尤其如此，干眼症直接影响泪膜及其成分。泪液蛋白质组学分析具有挑战性，主要是由于蛋白质的动态范围很广，而且样本量很小。然而，近年来质谱技术的进步彻底改变了蛋白质组学领域，即使使用小样本量，也能实现前所未有的深度、速度和准确性。在本研究中，我们使用 Orbitrap Fusion Tribrid 质谱仪，比较了四种不同的质谱工作流程，用于通过 Schirmer 条收集的泪液的蛋白质组学分析。我们能够建立一种在条带上进行蛋白质消化的方法，该方法能够在 11 名健康受试者的人泪样本中鉴定出超过 3000 种蛋白质。与以前不进行样品混合的报道方法相比，我们的方法在鉴定的蛋白质数量上有了显著的提高。
