High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

Citrullination is a post-translational modification implicated in autoimmune and inflammatory diseases, yet its low abundance and lack of effective enrichment tools have limited proteome-wide analysis. Here, we developed a robust chemical proteomics workflow with improved specificity and throughput. This method builds upon glyoxal-based derivatization and incorporates a cleavable biotin linker for efficient peptide enrichment, release, and identification via mass spectrometry. Benchmarking across biological systems demonstrated a >10-fold increase in the detection of citrullinated peptides (> 150-fold increase in intensity) at sub-0.1% abundance. Applying the workflow to mouse brain tissue and human primary neutrophils revealed dynamics and condition-specific changes in the citrullinome, including previously uncharacterized sites and regulatory processes. Notably, extensive citrullination of linker histone H1 and structural proteins such as lamin B1 in ionomycin-activated neutrophils suggests broad remodeling of cell architecture via citrullination. This workflow enables proteome-wide mapping of citrullination sites and facilitates its study across diverse biological contexts.