Multiplex autoantibody profiling using 'synovial proteome' microarrays identifies citrulline-modified peptides as major targets of the autoimmune response in rheumatoid arthritis

Introduction

Proteomics approaches to comprehensively characterize the spectrum of B-cell autoreactivity in autoimmune diseases are an exciting new area of research. We have developed autoantigen microarrays for multiplex autoantibody profiling, and have previously demonstrated specific and sensitive detection of autoantibodies in a variety of autoimmune diseases, including systemic lupus erythematosus, Sjögren's disease and mixed connective tissue disease [1]. Rheumatoid arthritis (RA) is an autoimmune disease affecting 0.8% of the world population, driven by as yet unidentified autoantigens. Recent research suggests that deimination of certain proteins gives rise to immunogenic epitopes that may play a key role in RA autoimmunity. This post-translational modification is characterized by the conversion of the amino acid arginine to citrulline, catalyzed by peptidyl arginine deiminase.

Objective

We use our 1344-feature 'synovial proteome' antigen microarrays to test the hypothesis that microarray-based specific detection of autoantibodies against candidate antigens enables diagnosis and classification of RA.

Methods

Our 1472-feature synovial proteome microarrays contain more than 300 distinct proteins and peptides, representing putative autoantigens in RA. Antigens include native and citrullinated fibrinogen, vimentin and keratin, citrulline-modified filaggrin and fibrinogen peptides, heterogeneous nuclear ribonucleoprotein (hnRNP)-B1, hnRNP-D, and overlapping hnRNP-A2 peptides, collagen type I, type II, type III, type IV and type V, overlapping human cartilage glycoprotein-39 peptides, several heat shock proteins and the endoplasmic reticulum molecular chaperone BiP, glucose-6-phosphate isomerase, acetyl-calpastatin, and many other antigens.

Results

Statistical algorithms demonstrated specific reactivity in RA sera against combinations of citrulline-modified filaggrin and fibrinogen peptides as well as deiminated proteins, but not against corresponding control peptides and proteins containing native arginine residues. Array analysis of two separate cohorts of RA patients and control patients for reactivity against panels of deiminated proteins and peptides provided a sensitivity of 45% at a specificity level of 95% for the diagnosis of RA. Reactivity against other antigens was observed but not specific.

Summary

The data suggest that multiplex autoantibody profiling allows for the specific and sensitive detection of autoantibodies directed against citrulline-modified filaggrin peptides and other deiminated polypeptides in RA. Array-based studies are underway to determine the fine specificity of autoimmune responses against panels of citrulline-modified peptides.