Untargeted Tear Proteomics in a Large South-Asian Cohort Reveals Inflammatory Signaling, ECM Remodeling, and Altered Metabolism in Keratoconus.

Purpose: Keratoconus (KC), a progressive corneal degenerative disease, is characterized by focal thinning and weakening, and the molecular pathways driving such changes are still being discovered. The progression-related pathologic molecular factors have not been identified in genetic studies from KC, and stage-specific molecular changes remain unknown in prior protein studies. We address this challenge through untargeted mass spectrometry analysis in a large KC cohort. Methods: The cohort comprised 40 healthy individuals and 107 eyes with varying KC grades from 69 individuals. Quantitative proteomics using iTRAQ labeling coupled with two-dimensional nanoLC-ESI-MS/MS (TripleTOF 5600) was employed followed by validation. Results: Unbiased LC-MS/MS analysis identified 1104 proteins, with 279 quantified proteins. Thirty-two proteins exhibited significant dysregulation in tear fluids compared to the control, enriched in glycolytic pathways, extra-cellular matrix (ECM) organization, reactive oxygen detoxification, and inflammatory regulation. Cystatin-S, lacritin, glutathione synthetase, and superoxide dismutase were validated to have differential expression across each KC grade. Conclusions: Our data unveiled novel tear fluid proteins involved in unique biological processes such as neutrophil degranulation, autophagy, metabolic alterations, protein phosphorylation, and more, apart from the ECM modulation and inflammatory pathways. Although the newly identified progressive KC biomarkers will help in disease characterization, identified molecular pathways may serve as novel therapeutic targets.