iTRAQ-based quantitative proteomic analysis of tear fluid in rat penetrating keratoplasty model with acute corneal allograft rejection

Abstract

 

Purpose

 

This study aims to develop further understanding of the mechanisms of acute corneal allograft rejection by identifying tear proteins at defined stage and to to discover potential interesting protein of corneal rejection.

 

Methods

 

iTRAQ-2DLC-MS/MS technique was used to uncover tear proteins that were changed in rat penetrating keratoplasty (PKP) model at different time points. Bioinformatics technology was applied in the analysis of significant proteins. The interesting protein was verified by western blotting.

 

Results

 

A total of 269 proteins were quantified, and 118 proteins were considered as significant proteins by at least 2.0-or 0.5- fold. For GO annotation, the top enrichments were neurological disease, free radical scavenging, cell death and survive and cell movement. For pathway analysis, the top enrichments were LXR/RXR activation, acute phase response signaling, clathrin-mediated endocytosis signaling and coagulation system. Coronin-1A was considered as an interesting protein for early stage of acute corneal allograft rejection.

 

Conclusions

 

This study first demonstrates that tear proteomics is a powerful tool for further understanding of the mechanisms of acute corneal rejection, and coronin-1A in tears might be closely related to allograft rejection.

 

 

GO annotation and pathway analysis from IPA database. A, top enrichments of functions of differential expressed proteins in allograft group; B, top enrichments of functions of differential expressed proteins in both allograft and autologous groups. C, top enrichments of pathways of differential expressed proteins in allograft group; D, top enrichments of pathways of differential expressed proteins in both allograft and autologous groups.

 

 

Identification and relative quantification of coronin-1A using iTRAQ. A, the expression levels of coronin-1A for 7 iTRAQ labeled samples; B, the sequences of peptide DAGPLLISLK, which form coronin-1A; C, another identified peptide sequences with KSDLFQEDLYPPTAGPDPALTAEEWLSGR leading to the identification of coronin-1A.

 

Keywords: 663 proteomics • 480 cornea: basic science • 555 immunomodulation/immunoregulation