Comparative proteomic analysis of tear fluid in Graves' disease with and without orbitopathy

The tears, a critical body fluid of the surface of the eye, contain an unknown number of molecules including proteins/peptides, lipids, small molecule metabolites, and electrolytes. There have been continued efforts for exploring the human tear proteome to develop biomarkers of disease. In this study, we used the high speed TripleTOF 5600 system as the platform to analyze the human tear proteome from healthy subjects (3 females and 1 male, average age: 36±14). We have identified 1543 proteins in the tears with less than 1% false discovery rate, which represents the largest number of human tear proteins reported to date. The data set was analyzed for gene ontology (GO) and compared with the human plasma proteome, NEIBank lacrimal gland gene dataset and NEIBank cornea gene dataset. This comprehensive tear protein list may serve as a reference list of human tear proteome for biomarker research of ocular diseases or establishment of MRM (Multiple Reaction Monitoring) assays for targeted analysis. Tear fluid is a useful and an accessible source not only for evaluating ocular surface tissues (cornea and conjunctiva), inflammation, lacrimal gland function and a number of disease conditions, such as dry eye as well as response to treatment. Copyright © 2012 Elsevier B.V. All rights reserved.

The proteins found in tears have an important role in the maintenance of the ocular surface and changes in the quality and quantity of tear components reflect changes in the health of the ocular surface. In this study, we have used quantitative proteomics, iTRAQ technology coupled with 2D-nanoLC-nano-ESI-MS/MS and with a statistical model to uncover proteins that are significantly and reliably changed in the tears of dry eye patients in an effort to reveal potential biomarker candidates. Fifty-six patients with dry eye and 40 healthy subjects were recruited for this study. In total, 93 tear proteins were identified with a ProtScore >or=2 (>or=99% confidence). Associated with dry eye were 6 up-regulated proteins, alpha-enolase, alpha-1-acid glycoprotein 1, S100 A8 (calgranulin A), S100 A9 (calgranulin B), S100 A4 and S100 A11 (calgizzarin) and 4 down-regulated proteins, prolactin-inducible protein (PIP), lipocalin-1, lactoferrin and lysozyme. Receiver operating curves (ROC) were evaluated for individual biomarker candidates and a biomarker panel. With the use of a 4-protein biomarker panel, the diagnostic accuracy for dry eye was 96% (sensitivity, 91.0%; specificity, 90.0%). Two biomarker candidates (alpha-enolase and S100 A4) generated from iTRAQ experiments were successfully verified using an ELISA assay. The levels of these 10 tear proteins reflect aqueous secretion deficiency by lacrimal gland, inflammatory status of the ocular surface. The clinical classification of the severity of the dry eye condition was successfully correlated to the proteomics by using three proteins that are associated with inflammation, alpha1-acid glycoprotein 1, S100 A8 and S100 A9. The nine tear protein biomarker candidates (except alpha1-acid glycoprotein 1) were also verified using an independent age-matched patient sample set. This study demonstrated that iTRAQ technology combined with 2D-nanoLC-nanoESI-MS/MS quantitative proteomics is a powerful tool for biomarker discovery.

Environmental, genetic, and immune factors are at play in the development of the variable clinical manifestations of Graves' ophthalmopathy (GO). Among the environmental contributions, smoking is the risk factor most consistently linked to the development or worsening of the disease. The close temporal relationship between the diagnoses of Graves' hyperthyroidism and GO have long suggested that these 2 autoimmune conditions may share pathophysiologic features. The finding that the thyrotropin receptor (TSHR) is expressed in orbital fibroblasts, the target cells in GO, supported the notion of a common autoantigen. Both cellular and humeral immunity directed against TSHR expressed on orbital fibroblasts likely initiate the disease process. Activation of helper T cells recognizing TSHR peptides and ligation of TSHR by TRAb lead to the secretion of inflammatory cytokines and chemokines, and enhanced hyaluronic acid (HA) production and adipogenesis. The resulting connective tissue remodeling results in varying degrees extraocular muscle enlargement and orbital fat expansion. A subset of orbital fibroblasts express CD34, are bone-marrow derived, and circulate as fibrocytes that infiltrate connective tissues at sites of injury or inflammation. As these express high levels of TSHR and are capable of producing copious cytokines and chemokines, they may represent an orbital fibroblast population that plays a central role in GO development. In addition to TSHR, orbital fibroblasts from patients with GO express high levels of IGF-1R. Recent studies suggest that these receptors engage in cross-talk induced by TSHR ligation to synergistically enhance TSHR signaling, HA production, and the secretion of inflammatory mediators.