The tear film is a thin (2-6 µm thick) multi-layer fluid that coats the corneal and conjunctival epithelia The mucoaqueous layer is the thickest layer of the tear film under normal physiological conditions whose direct interaction with the mucosal surface nourishes and protects the underlying corneal and conjunctival epithelial cells. The lipid layer, the outermost layer derived from the Meibomian glands, limits evaporation. The proximity of the tear film to the ocular milieu, the non-invasive nature of its collection, and its high protein concentration make it an attractive source for proteomic biomarker studies. Further, identifying the protein contents of tear fluid is vital in understanding their physiological and pathological roles in the eye.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as the analytical method of choice because of its high-throughput nature, sensitivity, high dynamic range, and ability to identify complex mixtures even from small sample volumes. However, in LC-MS/MS profiles, the proteins with high concentrations have a higher chance of detection, whereas the proteins with lower concentrations are detected only in a smaller percentage of samples due to random chance. It is exceedingly difficult to draw statistical conclusions for the proteins with a very low detection rate and should be excluded at the data analysis step. However, making such decisions based on smaller sample sets can lead to poor reproducibility. Therefore, a reference list of tear proteins detected reliably using a larger sample set may be helpful in alleviating these concerns.