Abstract
PURPOSE: Thyroid eye disease (TED) is the most common extrathyroidal manifestation of Graves' disease (GD). Despite its clinical significance, the pathogenic mechanisms and reliable diagnostic biomarkers for TED remain incompletely defined. Tear fluid offers a noninvasive window into disease-related molecular changes. METHODS: Tear samples were collected using Schirmer strips from 20 patients with TED, 20 patients with GD without ocular involvement, and 14 healthy controls (HCs). Proteomic profiling was performed using a novel pressure cycling technology-pulse data-independent acquisition mass spectrometry (PCT-PulseDIA-MS) workflow. RESULTS: A total of 5966 tear proteins were quantified. Differentially expressed proteins (DEPs) were identified through pairwise group comparisons. Patients with TED showed the most extensive tear proteomic alterations among the studied groups. One hundred seventy-four DEPs were associated with ophthalmopathy, 14 with autoimmunity, and 13 with hyperthyroidism. The ophthalmopathy-related DEPs were enriched in immune regulation, lipid metabolism, vascular function, and extracellular matrix remodeling. Several key DEPs showed significant correlations with clinical, laboratory, and imaging variables. A three-protein panel comprising calcium-activated nucleotidase 1 (CANT1), insulin-like growth factor-binding protein 7 (IGFBP7), and caspase 14 (CASP14) achieved excellent diagnostic performance in distinguishing TED from GD, with an area under the curve (AUC) of 0.971. CONCLUSIONS: Tear proteomics reveals distinct molecular signatures shaped by the combined influences of ophthalmopathy, autoimmunity, and hyperthyroidism throughout the pathogenesis of TED, underscoring the potential of tear proteins as early, noninvasive biomarkers for disease diagnosis.