Unraveling the Intraday Variations in the Tear Fluid Proteome.

Purpose: Tear fluid is a complex and dynamic biological fluid that plays essential roles in maintaining ocular homeostasis and protecting against the external environment. Owing to the small sample volume, studying the tear proteome is challenging. However, advances in high-resolution mass spectrometry have expanded tear proteome profiling, revealing >500 unique proteins. Tears are emerging as a noninvasive source of biomarkers for both ocular and systemic diseases; nevertheless, intraday variability of proteins in tear fluid remains questionable. This study investigates intraday variations in the tear fluid proteome to identify stable proteins that could act as candidate biomarkers. Methods: Tear samples from 15 individuals at four time points (10 am, 12 pm, 2 pm, and 4 pm) were analyzed using mass spectrometry to evaluate protein variation during these intervals. Technical variation was assessed by analyzing pooled samples and was subtracted from the total variation to isolate biological variability. Results: Owing to high technical variation, low-abundant proteins were filtered, and only 115 proteins met the criteria for further analysis. These criteria include being detected at all four time points in at least eight subjects, having a mean peptide-spectrum match count greater than 5, and having a technical variation less than 0.10. Lactotransferrin, lipocalin-1, and several immunoglobulins were among the 51 stable proteins (mean biological coefficient of variation < 0.10). Additionally, 43 proteins displayed significant slopes across the 4 time points, with 17 increasing and 26 decreasing over time. Conclusions: These findings contribute to the understanding of tear fluid dynamics and further expand our knowledge of the tear proteome.