Precision in Tear Fluid Biomarker Discovery: Quantitative Proteomic Profiling of Small-Volume, Individual Samples Using Capillary Tube Collection

Background/Objectives: Tear fluid, a complex biofluid that contains thousands of proteins and can be collected non-invasively, has emerged as a promising source of biomarkers for ocular and systemic health. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is currently the primary method for discovering novel biomarkers in tear fluid. However, the method of tear collection can significantly impact LC-MS/MS analysis outcomes. Tear fluid is commonly collected using either Schirmer strips or capillary tubes. While capillary tubes offer distinct advantages, such as reduced extracellular contamination and reflex tearing, most LC-MS/MS protocol development has focused on optimizing protocols for Schirmer strips. This study addresses this gap by evaluating digestion protocols for tear fluid collected with capillary tubes, focusing on biomarker discovery using small-volume samples. In this study, we evaluated multiple digestion protocols for the shotgun quantitative LC-MS/MS analysis of small-volume tear fluid samples collected using glass capillary tubes. Protocol optimization was performed using pooled samples and then compared with the analysis of individual samples. With the optimized protocol, we identified an average of 368 ± 87 proteins in pooled samples and 502 ± 127 proteins in individual small-volume tear fluid samples, using less than 1µL of total fluid volume. This protocol highlights the practicality of using glass capillary tubes for comprehensive LC-MS/MS-based tear proteomics analysis, paving the way for detailed proteomics characterization of individual tear fluid samples rather than pooled samples. By shifting from pooled to individual samples, this approach greatly accelerates tear biomarker discovery, advancing precision and personalized medicine.