Deciphering Membrane-Associated Molecular Processes in Target Tissue of Autoimmune Uveitis by Label-Free Quantitative Mass Spectrometry

Abstract

Purpose: : Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Although retinal-autoantigen specific T-helper 1 cells have been demonstrated to trigger disease progression and relapses, the molecular processes leading to retinal degeneration and consequent blindness remain unknown. In order to identify molecular pathways involved in disease pathomechanisms, we quantitatively compared protein expressions in membrane-enriched fractions of ERU target tissue retina with healthy control tissue.

Methods: : Membrane proteins from total retinal extracts were enriched by step-wise extraction method. Resulting protein pools from healthy and ERU-diseased tissue were then subject to tryptic digestion and analysed by liquid chromatography mass spectrometry (LC-MSMS, OrbiTrap). All detected peptide features (>20000) were aligned and statistically analysed for differential abundance (cumulated peak intensities). Peptides were identified by database search (Ensembl, species equus caballus) and resulting protein IDs were grouped according to their mode of regulation. Pathway enrichment analyses (KEGG spider, Consensus PathDB) were used for identification of overrepresented pathways. Candidate proteins were validated by quantitative immunohistochemistry on a large collection of diseased tissue samples.

Results: : Quantitative comparisons of membrane-enriched tissue fractions resulted in a total identification of 893 proteins of which approximately 30% were membrane proteins. Of these, 164 proteins were downregulated (p<0.05, ≥2fold) in ERU and 175 proteins were found upregulated (p<0.05, ≥2fold) in ERU. Pathway enrichment analyses with these regulated proteins resulted in the identification of several significantly overrepresented pathways, among them "antigen processing and presentation" (BioCarta), "Integrin cell surface interactions " (Reactome) and "Focal adhesion" (KEGG). Validation of candidate proteins from those pathways by quantitative immunohistochemistry on tissue specimen (n=10) confirmed the regulation.

Conclusions: : The new analytical strategy increases the analytical depth as compared to the classical 2D gel approach raising the chance for discovery of clinically relevant biomarkers as well opening the door towards detection of membrane-associated pathological processes triggering the onset of the disease.

Keywords: autoimmune disease • proteomics • retinal degenerations: cell biology