Precision in Tear Fluid Biomarker Discovery: Quantitative Proteomic Profiling of Small-Volume, Individual Samples...

Background: Tear fluid, rich in proteins, is a promising source of novel biomarkers for ocular and systemic health. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the primary method for biomarker discovery. Still, factors such as limited sample volume, extracellular protein contamination, and reflex tearing can significantly impact results. Glass microcapillary tubes minimize these issues. Schirmer strips remain the most common collection method due to existing LC-MS/MS protocol optimization. Methods: 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. Results: Using the optimized protocol, one μL samples were processed using a timsTOF Pro 2 mass spectrometer (Bruker) coupled online with an Evosep One liquid chromatography system (Evosep), leading to the identification of an average of 361 ± 63 proteins in pooled samples and 525 ± 123 proteins in individual small-volume tear fluid samples. Conclusions: 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.