Standardizing tear proteomics preanalytics: Capillary vs Schirmer sampling and denaturing vs mild elution buffers...

Tear fluid is a promising minimally invasive source of biomarkers for ocular surface diseases (OSDs). However, variability in sampling and pre-analytical processing remains a major limitation for reproducibility and cross-study comparability. Here, we quantitatively evaluated two commonly used tear collection methods, microcapillary tubes (CT) and Schirmer strips (SCH), and assessed the effect of elution buffer composition on protein recovery from SCH. In Study I, tears were collected from the same healthy donors using CT and SCH. In Study II, SCH from additional donors were longitudinally split and eluted with phosphate-buffered saline (PBS) or a denaturing cell lysis buffer (CLB; 7 M urea, 2 M thiourea, 4% CHAPS), enabling paired within-strip comparisons. Samples were analyzed by Evosep-timsTOF Pro DIA mass spectrometry and quantified using library-free DIA-NN. Overall, 3749 proteins were identified. SCH markedly increased proteome coverage compared with CT, reflecting recovery of both soluble tear proteins and ocular surface-derived cellular material. Functional annotation showed enrichment of intracellular compartments in SCH samples, particularly cytoplasmic, nuclear, and cytoskeleton-related proteins. For SCH extraction, CLB modestly increased unique protein identifications compared with PBS while preserving high quantitative concordance. These findings show that pre-analytical choices strongly influence tear proteome depth and abundance profiles. SIGNIFICANCE: Tear proteomics is increasingly recognized as a valuable platform for biomarker discovery in ocular, neurological, and systemic diseases. However, substantial methodological heterogeneity in tear collection and pre-analytical processing remains a major source of variability, limiting reproducibility and cross-study comparability. By directly comparing microcapillary tube and Schirmer strip sampling within the same donors, and by evaluating mild versus denaturing elution conditions from Schirmer strips using a within-strip paired design, this study provides quantitative evidence that pre-analytical choices systematically shape both proteome depth and quantitative abundance profiles. Schirmer strip sampling markedly expands detectable proteome coverage, partly through co-recovery of ocular surface-derived cellular material, whereas denaturing extraction enhances the recovery of protein subsets insufficiently solubilized under mild conditions while preserving high quantitative concordance. Collectively, these findings demonstrate that sampling and extraction strategies are not neutral technical variables but key determinants of the biological space interrogated by tear proteomics. The data presented here inform rational study design, improve inter-laboratory comparability, and support the development of harmonized standard operating procedures for robust tear-based proteomic biomarker discovery and translational applications.