Development of a Chemoproteomic Platform to Identify Sites of (Homo)citrullination within Complex Proteomes

Le résumé fourni par la source

Citrullination and homocitrullination of arginine and lysine can significantly impact protein structure and function. Citrullination of arginine is an enzymatic modification catalyzed by Protein Arginine Deiminases (PADs). Homocitrullination of lysine is a nonenzymatic modification that occurs in the presence of high concentrations of cyanate. Both post-translational modifications are elevated in Rheumatoid Arthritis (RA) and other inflammatory diseases. Moreover, autoantibodies targeting these PTMs are associated with the development of RA. Identifying arginine and lysine residues that are hypersensitive to these modifications is critical for deepening our understanding of the functional effects of (homo)citrullination. Current methods use a phenylglyoxal-biotin probe for the protein-level identification of citrullinated proteins, however, this platform does not inform on the exact site of citrullination. Herein we describe the development of a desthiobiotin-phenylglyoxal (DB-PG) probe, which can be used to selectively enrich and subsequently release (homo)citrullinated peptides for the site-specific identification of citrullinated arginines and homocitrullinated lysines. (Homo)citrullinated peptides enriched using DB-PG were subjected to quantitative mass-spectrometry analysis to (1) identify PAD2 and PAD4-selective citrullination sites across ∼800 arginine residues and (2) evaluate ∼1400 lysine residues for sensitivity to homocitrullination by cyanate. Projecting forward, this platform will enable the comprehensive analysis of (homo)citrullination in complex proteomes.

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