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

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.