High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

Meelker Gonzalez, R.; Laposchan, S.; Riedel, E.; Fürst, A.; O'Sullivan, N.; Gabriel, W.; Wilhelm, M.; Knolle, P. A.; Medard, G.; Kuster, B.; Lee, C.-Y.

doi:10.1101/2025.04.25.650601

bioRxiv · 10.1101/2025.04.25.650601

High-throughput chemical proteomics workflow for profiling protein citrullination dynamics

Meelker Gonzalez, R.·Laposchan, S.·Riedel, E.·Fürst, A.·O'Sullivan, N.·Gabriel, W.·Wilhelm, M.·Knolle, P. A.·Medard, G.·Kuster, B.·Lee, C.-Y.

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Abstract

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.

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Meelker Gonzalez, R., Laposchan, S., Riedel, E., Fürst, A., O'Sullivan, N., Gabriel, W., Wilhelm, M., Knolle, P. A., Medard, G., Kuster, B., Lee, C.-Y.. 2025-04-27. High-throughput chemical proteomics workflow for profiling protein citrullination dynamics. https://doi.org/10.1101/2025.04.25.650601

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