Chemical biology approaches for detecting and profiling protein citrullination

The development of citrullination detection methods has largely been driven by a fundamental chemical problem: distinguishing citrulline from its precursor arginine. Protein citrullination is a biologically relevant modification catalyzed by the peptidyl arginine deiminase (PAD) enzyme family. Dysregulated PAD activity has been implicated in several major human diseases, including rheumatoid arthritis, neurodegeneration, and cancer. In practice, studying this modification has long been difficult because the two amino acids, citrulline and arginine, are so chemically similar that most chemical reagents struggle to distinguish between them. This review highlights the evolution of citrullination detection, from colourimetric assays requiring harsh acidic conditions, through fluorescent probes that increased sensitivity, and to newer chemistries that can label citrulline at neutral pH. Alongside these citrulline-directed strategies, we also cover complementary approaches for profiling PAD enzyme activity, including antibody-based and activity-based protein profiling, as well as fluorogenic substrate assays.