On-Array Citrullination of Protein Microarrays

Protein microarrays are a powerful tool for investigating autoantigens that drive autoimmune diseases. However, in many cases, autoantibody reactivity is directed against slightly modified proteins, generating neo-autoantigens, rather than targeting native antigens. Citrullination, a post-translational modification, is a key example, playing a central role in the pathogenesis of rheumatoid arthritis (RA). Excessive activity of peptidylarginine deiminases (PAD) leads to the formation of neo-autoantigens that trigger an autoimmune response. This chapter describes a detailed protocol for on-array citrullination of protein microarrays. By incubating the arrays with a PAD-containing buffer, the protocol attempts to replicate the formation of citrullinated autoantigens that occurs in vivo and contributes to RA. This approach enables high-throughput identification of citrullinated autoantigens, providing a platform to identify novel autoantigens and explore their role in RA pathology.