Abstract
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.
Highlights
Published: 19 February 2022The tear film is a 2–6 μm stratified fluid, composed of mucoaqueous and lipid layers, that covers the corneal and conjunctival epithelia of the ocular surface
While our in-strip protein digestion method produced a clear improvement in the number of identified proteins and peptides compared to pre-extraction digestion, both collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD)
Given the abundance of glycoproteins in tears [23], as well as the significant roles they play in maintaining ocular surface homeostasis [24], HCD fragmentation combined with in-strip digestion was selected as the optimal workflow for the proteomic analysis of tears; this will facilitate future glycoproteomic studies
Summary
The tear film is a 2–6 μm stratified fluid, composed of mucoaqueous and lipid layers, that covers the corneal and conjunctival epithelia of the ocular surface. The outermost layer of the tear film is a thin lipid layer that prevents evaporation of the mucoaqueous layer [2,3]. Proteins and lipids of the tear film safeguard the eye through anti-inflammatory, antioxidant, and antimicrobial activities [4,5,6,7]. Insufficient secretion of the aqueous or lipid components of tears, laser eye surgery, and inflammatory pathologies, such as Sjögren’s syndrome, can result in tear film disruption, leaving the eye more vulnerable to infection and inflammation [8,9]. The integrity of this film is necessary for maintaining normal vision and preventing ocular damage
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