Specific modification of peptide-bound citrulline residues

Immune reactions to citrulline-containing proteins appear to be central in the immunopathogenesis of rheumatoid arthritis. Citrulline residues are introduced into proteins by deimination of arginine residues, likely by an enzymatic process. There is a need to characterize which proteins in the inflamed joints of rheumatoid patients contain citrulline in situ. The characterization of deiminated proteins will be greatly facilitated by specific modification of peptide-bound citrulline residues that will enable specific enrichment and detection of citrulline-containing peptides. This study presents the details of such a modification method. The chemistry behind the reaction of the ureido group of citrulline with 2,3-butanedione in the presence of antipyrine is unraveled. Parameters for optimization of the reaction with respect to specificity and completeness, including the testing of different acids, reactant concentrations, and reaction time, are presented. This modification reaction is specific for citrulline residues. The modified product shows a characteristic mass shift of +238 Da, as demonstrated by mass spectrometry. The product absorbs UV–Vis radiation at 464 nm, and it is demonstrated that this can be used to selectively monitor citrulline-containing peptides during the separation of protein digests. Finally, the structure of the product of modified citrulline is solved by nuclear magnetic resonance spectroscopy using N-butylurea as a model substance. The results presented should facilitate the development of tags that can be used for the enrichment and subsequent detection of citrulline-containing protein fragments by mass spectrometry.