Sophia Laposchan

Protein arginine deiminases (PADs) convert arginine to citrulline, altering protein structure. Of the five human isozymes, PAD1–4 are catalytically active with distinct tissue-specificities, yet isozyme-specific substrate recognition remains poorly defined. Here, we perform in vitro lysate-based substrate profiling of PAD1–4 via mass spectrometry across human H4 and HeLa cell lines, identifying ~30,000 citrullination sites across ~5,500 proteins. Only 14% of sites are shared among all, reflecting distinct sequence preferences: PAD1–2 show broad specificity, whereas PAD3–4 favor arginines flanked by acidic or glycine residues. These preferences persist over 10 min–16 h, indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants reveal Q346, G403, R639, and H640 as key determinants distinguishing substrate recognition from that of PAD2. This work provides a comprehensive in vitro atlas of maximal substrate capacity, defining isozyme-specific motifs and molecular determinants to guide selective inhibitors and probes for citrullination mechanisms in health and disease.

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 develop 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 demonstrates a > 10-fold increase in the detection of citrullinated peptides at sub-0.1% abundance. Applying this workflow to primary human neutrophils, we successfully monitor dynamic regulation, quantifying dose-dependent activation and inhibition by the PAD4 inhibitor GSK484. Furthermore, stimulation with the fungal pathogen Candida albicans reveals a “core citrullinome” conserved across distinct stimuli. Notably, extensive citrullination of linker histone H1 and structural proteins like lamin B1 suggests broad remodeling of cell architecture during NET formation. This workflow enables proteome-wide mapping of citrullination sites and facilitates its study across diverse biological contexts.

Protein arginine deiminases (PADs) convert arginine to citrulline, altering protein structure and function. Of the five human isozymes, PAD1–4 are catalytically active with distinct tissue-specificities, yet isozyme-specific substrate recognition remains poorly defined. We profiled PAD1–4 substrate landscapes via mass spectrometry, identifying ∼30,000 citrullination sites across ∼5,500 proteins. Only 14% of sites were shared among all four, reflecting distinct sequence preferences: PAD1–2 showed broad specificity, whereas PAD3–4 favored arginines flanked by acidic or glycine residues. These preferences persisted over 10 min–16 h, indicating sequence context rather than temporal dynamics drives specificity. Mutation analysis of eleven PAD4 variants revealed Q346, G403, R639, and H640 as key determinants distinguishing substrate recognition from that of PAD2. This work provides the most comprehensive PADs substrate atlas to date, defining isozyme-specific motifs and molecular determinants, and guiding development of selective inhibitors and probes to interrogate citrullination mechanisms in health and disease.

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.

Protein citrullination, a post-translational modification (PTM) catalyzed by peptidylarginine deiminases (PADs), plays critical roles in biological processes such as immunity, gene regulation, and inflammation. Dysregulated citrullination is implicated in diseases including rheumatoid arthritis, multiple sclerosis, and cancer, making it a potential biomarker and therapeutic target. Immunodetection is the most commonly used technique to study citrullination. However, most commercially available antibodies against citrullination are either protein-specific or lack the sensitivity and specificity of the broad variety of modified proteins. In addition, existing anti-pan citrullination antibodies often fail to distinguish citrullination from homocitrullination, a chemically similar modification on lysine. This cross-reactivity limits their utility in deciphering the distinct biological roles of these PTMs. To address these challenges, we employed a motif-based strategy to generate monoclonal antibodies against citrullination. This approach leverages PAD enzyme sequence preferences from human proteome, enhancing specificity for citrullination while minimizing cross-reactivity with homocitrullination. We immunized rats with a pool of over 490,000 citrullinated peptides, designed to represent common citrullination motifs in human tissue proteomes. Two monoclonal antibody clones were established and validated for sensitivity and specificity using ELISA and western blot against in vitro citrullinated and homocitrullinated proteomes, as well as ionomycin-activated human neutrophils. Both clones demonstrated great sensitivity to diverse citrullinated proteins, robust discrimination against homocitrullination, and quantitative readout in biological samples. These novel antibodies provide powerful tools for studying global citrullination dynamics and hold promise for biomarker discovery and diagnostic applications in diseases involving PAD dysregulation.