Label-free LC-MSMS-based Differential Proteome Analysis of Vitreous from Autoimmune Uveitis Cases

Abstract

Purpose: : Equine recurrent uveitis (ERU) is a devastating immune mediated inflammation targeting the inner eye and ultimately leading to blindness in the affected animals. The widespread disease (10%) is characterized by recurring episodes of inflammation followed by quiescent stages. ERU represents the only spontaneous animal model for human autoimmune uveitis and thus serves as a valuable model for exploration of as yet unknown pathomechanisms relevant for disease progression and relapses. Since vitreous is directly adjacent to the inflamed retina this compartment facilitates indirect exploration of ongoing disease related events in the retina. To investigate protein expression changes related to the disease, we undertook a comprehensive differential proteome profiling of ERU and healthy vitreous.

Methods: : Proteins from vitreous samples of healthy horses and ERU cases (n=16) were subjected to tryptic digestion and analysed by liquid chromatography mass spectrometry (LC-MSMS; OrbiTrap). All detected peptide features were aligned and statistically analysed for differential abundance based on cumulated peak intensities. Peptides were identified by database search (Ensembl, Equus Caballus) and resulting protein IDs were grouped according to their mode of regulation. Pathway enrichment analyses (Consensus PathDB and Genomatix Pathway Systems) were used for identification of overrepresented pathways per condition. Candidate proteins were validated by western blots on a large sample collection.

Results: : Highly sensitive comparisons of vitreous samples resulted in identification of 251 proteins, 105 of these were identified with very high confidence (2 or more peptides, p<0.01). 26 proteins were found upregulated in ERU cases, while 44 were downregulated. Pathway enrichment analyses with these regulated proteins resulted in the identification of several significantly overrepresented pathways, among them "ECM-receptor interaction" (KEGG) and "Wnt signaling" (INOH). From these pathways, selected candidates were validated by western blots and mode of regulation observed by quantitative mass spectrometry was confirmed in all cases. Interaction network analyses based in literature mining enabled to suggest potential pathomechanisms and will be discussed in detail.

Conclusions: : Quantitative label-free proteomics based on LC-MSMS identified a signature of protein markers in the vitreous of uveitic eyes that point to dynamic changes in both extracellular matrix homeostasis and wnt signalling. As both molecular networks are mechanistically linked to inflammation as well as vascular changes in the eye, members of these protein networks are likely involved in the pathogenesis of uveitis.

Keywords: proteomics • autoimmune disease • Muller cells