Tear specific potential protein biomarker identification by 2D-DIGE based proteomics in Dry eye syndrome associated with Rheumatoid Arthritis

Abstract

Purpose: Dry eye syndrome (DES) is a disorder of the tear and ocular surface. The tear film dynamics maintain normal corneal function. Sjogrens syndrome (SS) secondary to Rhematoid Arthritis (RA) is an autoimmune disease affecting the extracellular glands like lacrimal, salivary and therefore DES is more prevalent in RA. This study aimed to identify potential biomarkers in tear in DES secondary to RA (RA-DES) based on 2D-based DIGE analysis compared to primary sjogrens (PS-DES), non sjogrens (NS-DES) and healthy controls.

Methods: As part of a prospective case-control study after IRB approval DES cases were recruited in the ocular surface clinic based on detailed dry eye work up and the report from rheumatoid clinic. Tear specimen were collected using schirmer strips from 102 DES cases (mean age: 45 ± 3 y) that included RA-DES, PS-DES, and NS-DES along with 56 age matched controls (mean age: 43 ± 12 y).Tear proteins were extracted using 8M urea buffer, subjected to 2D-Differential gel electrophoresis (DIGE). While pooled sample analysis was done by 2D-DIGE, independent samples were run by 2D. Differentially expressed proteins showing more than 2 fold variation were identified based on biological variance analysis (BVA) and extended discriminate analysis (EDA) using Decyder software. These peptide spots were then detected by nano ESI-LC-MS/MS analysis. Functional annotation was done using DAVID annotation tool.

Results: Of the 5 down regulated proteins in DES-RA, Lactotransferrin isoform 1 precursor showed down regulation in all the cases, SHC transforming 1 isoform in 63% cases, and the rest namely Ribonuclease protein subunit 20, Protocadherin and heterogeneous nuclear ribonucleoprotein Q isoform 6 showed down regulation in more than 80% cases. Three proteins were up regulated namely, Ecto-ADP ribosyltranferase 5 precursor, Rho-related GTP-binding protein and RhoJ precursor as seen in 80% cases. Functional annotation of proteins specific to DES-RA, revealed that these proteins have roles in regulation, antimicrobial activity, immune, metabolic and cellular processes.

Conclusions: The study revealed characteristic proteins that are differentially expressed in the tears of sjogrens syndrome secondary to RA. Further validation based on clinical correlations has to be done before characterising them as biomarkers.

Keywords: 486 cornea: tears/tear film/dry eye • 663 proteomics