Abstract
We analyzed the tear film proteome of patients with dry eye (DE), meibomian gland dysfunction (MGD), and normal volunteers (CT). Tear samples were collected from 70 individuals. Of these, 37 samples were analyzed using spectral-counting-based LC-MS/MS label-free quantitation, and 33 samples were evaluated in the validation of candidate biomarkers employing customized antibody microarray assays. Comparative analysis of tear protein profiles revealed differences in the expression levels of 26 proteins, including protein S100A6, annexin A1, cystatin-S, thioredoxin, phospholipase A2, antileukoproteinase, and lactoperoxidase. Antibody microarray validation of CST4, S100A6, and MMP9 confirmed the accuracy of previously reported ELISA assays, with an area under ROC curve (AUC) of 87.5%. Clinical endpoint analysis showed a good correlation between biomarker concentrations and clinical parameters. In conclusion, different sets of proteins differentiate between the groups. Apolipoprotein D, S100A6, S100A8, and ceruloplasmin discriminate best between the DE and CT groups. The differences between antileukoproteinase, phospholipase A2, and lactoperoxidase levels allow the distinction between MGD and DE, and the changes in the levels of annexin A1, clusterin, and alpha-1-acid glycoprotein 1, between MGD and CT groups. The functional network analysis revealed the main biological processes that should be examined to identify new candidate biomarkers and therapeutic targets.
Highlights
dry eye (DE) and meibomian gland dysfunction (MGD) diseases involve the disruption of the lacrimal functional unit, resulting in symptoms of discomfort and visual disturbance and tear film instability
We used the symptoms as a screening tool (OSDI), tear volume assessment to ascertain the subtype classification of DE (Schirmer test), and damage to the ocular surface as a standard test to study squamous metaplasia and goblet cell density of the conjunctiva
The following proteins were mostly overexpressed in DE: C3, S100A6, S100A8, CP, apolipoprotein D (APOD), ORM2, TXN, IGHG1, PLA2G2A, SERPINA1, and SLPI
Summary
DE and MGD diseases involve the disruption of the lacrimal functional unit, resulting in symptoms of discomfort and visual disturbance and tear film instability These illnesses may exist independently as either symptomatic or asymptomatic disorder; they are frequently found in the same patient. Conflicting results have been reported for other proteins such as secretoglobin 2A28,26,28 and serum albumin[14,20,26,27,28] These discrepancies might have been caused by several factors such as different sample collection methods or sensitivity and dynamic range of the employed quantitation techniques[29,30]. More extensive tear proteome studies focusing on systems biology, supported by orthogonal validation assays, are needed to extract meta-information common to all these studies The results of such studies should reduce the technological and experimental bias and improve our knowledge of the role of relevant proteins in the pathophysiology of ocular surface-related disorders. We have previously characterized tear film proteomes and identified biomarkers for the aqueous-deficient dry eye (ADDE) and MGD, the main cause of evaporative DE (EDE) condition[31], using 2D gel-based proteomics[24]
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