Protocol for identification of protein citrullination by immunoprecipitation followed by mass spectrometry

Protein citrullination occurs through the deimination of peptidyl-arginine to yield peptidyl-citrulline by one of the peptidyl-arginine deiminase (PAD) family members. This protocol identifies citrullinated protein residues using immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for identification of citrullination modifications in vitro or in cell culture, immunoprecipitation of nuclear citrullinated proteins, and identification of citrullinated residues by mass spectrometry (MS). This protocol applies to both recombinant protein assays and in vitro cell culture assays. For complete details on the use and execution of this protocol, please refer to Indeglia et al. 1 • Practical guidance for enriching citrullinated proteins • Optimized steps for isolating and immunoprecipitating large-scale nuclear extracts • Mass spectrometry-based workflow for confident identification of citrullination sites Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Protein citrullination occurs through the deimination of peptidyl-arginine to yield peptidyl-citrulline by one of the peptidyl-arginine deiminase (PAD) family members. This protocol identifies citrullinated protein residues using immunoprecipitation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We describe steps for identification of citrullination modifications in vitro or in cell culture, immunoprecipitation of nuclear citrullinated proteins, and identification of citrullinated residues by mass spectrometry (MS). This protocol applies to both recombinant protein assays and in vitro cell culture assays.