Skallerup Jacob, Poulsen Thomas B G, Stensballe Allan
Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.
Clinical Cancer Research Center, Aalborg University Hospital, Aalborg, Denmark.
Methods Mol Biol. 2025;2929:43-52. doi: 10.1007/978-1-0716-4595-6_4.
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.
蛋白质微阵列是研究引发自身免疫性疾病的自身抗原的强大工具。然而，在许多情况下，自身抗体反应针对的是轻微修饰的蛋白质，从而产生新自身抗原，而非靶向天然抗原。瓜氨酸化作为一种翻译后修饰，就是一个关键例子，在类风湿性关节炎（RA）的发病机制中起着核心作用。肽基精氨酸脱氨酶（PAD）的过度活性会导致新自身抗原的形成，进而引发自身免疫反应。本章描述了一种在蛋白质微阵列上进行瓜氨酸化的详细方案。通过将阵列与含PAD的缓冲液孵育，该方案试图重现体内发生的瓜氨酸化自身抗原的形成过程，这一过程与RA的发病有关。这种方法能够高通量鉴定瓜氨酸化自身抗原，为识别新型自身抗原并探索它们在RA病理中的作用提供了一个平台。
