High-Throughput Tear Proteomics via In-Capillary Digestion for Biomarker Discovery.

Tear fluid has emerged as a valuable resource for biomarker discovery; however, the limited sample volume, the dynamic composition, and the variability introduced by collection methods all present significant challenges to the analysis and interpretation of the results. A majority of tear proteomic studies have utilized Schirmer strips for tear fluid collection; however, microcapillary collection can provide a superior collection method for proteomic studies when analysis procedures are optimized. We developed a novel, high-throughput in-capillary trypsin digestion workflow that requires as little as 0.5 μL of tear fluid for bottom-up shotgun proteomics. The use of a single microcentrifuge tube for both tear collection and sample processing simplifies sample handling and minimizes both the sample loss and experimental errors associated with sample transfers. This streamlined approach also reduces sample processing time to under 2 h before overnight trypsin digestion, compared to the 5-8 h required by the other methods. Our method uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify more proteins with greater efficiency than the existing techniques. With this workflow, we identified 500-800 proteins per 0.5 μL sample without peptide fractionation, allowing for at least three technical replicates. The results showed a four-fold increase in the number of proteins identified in the samples. This approach validates the use of microcapillary tear collection, and the innovative processing technique significantly increases the throughput of tear proteomics for biomarker discovery.