Mass spectrometry protocol for tear fluid proteome profiling and biomarker discovery.

Here, we present a protocol for tear fluid proteomics using Schirmer strips and liquid chromatography-tandem mass spectrometry (LC-MS/MS). It includes tear sampling from human individuals and mice, followed by protein digestion, peptide desalting, and purification via solid-phase extraction. Supporting both data-dependent acquisition (DDA) and data-independent acquisition (DIA), this 5-h workflow maximizes protein recovery, minimizes contaminants, and enables reproducible biomarker discovery for ocular, neurodegenerative, and systemic diseases. Tear fluid's non-invasive collection and rich proteome make it ideal for clinical proteomics and personalized medicine.