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 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.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org)69 via the MassIVE partner repository with the dataset identifier MSV000097617 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?accession= MSV000097617]. All processed data generated in this study are provided in the Supplementary Information, Supplementary Data, and Source Data file. Source data are provided with this paper.
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Acknowledgements
The authors would like to thank Dr. Christina Ludwig, Dr. Andreas Zellner, Dr. Polina Prokofeva, Ms. Susanne Wudy, Mr. Genc Haljiti, Prof. Dr. Thomas Skurk, Dr. Kurt Rack, and members from the Bavarian Center for Biomolecular Mass Spectrometry (BayBioMS) and the Chair of Proteomics and Bioanalytics for their valuable assistance and insightful discussions. This work was funded by the German Federal Ministry of Education and Research: FKZ031L0215 (YIG-SysNS; C.Y.L.); FKZ161L0214A (CLINSPECT-M; B.K.); FKZ03LW0243K (CLINSPECT-M-2; B.K.) and ERC Starting Grant (grant number 101077037; M.W.). The Orbitrap Fusion Lumos and Orbitrap Astral mass spectrometers used in this study were funded in part by the German Research Foundation (DFG-INST 95/1436-1 FUGG & DFG-INST 95/1859-1 FUGG). The graphical illustrations were created with BioRender.com.
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G.M., B.K., and C.Y.L. conceptualized the study. R.M.G. and C.Y.L. designed experiments. R.M.G. performed experiments on development of the methodology. R.M.G., S.L., E.R., A.F., and N.O. performed experiments on biological samples. R.M.G., S.L., W.G., and C.Y.L. analyzed the data. M.W., P.A.K., and B.K. provided analysis tools, animal samples, and instrumentation. R.M.G. and C.Y.L. wrote the manuscript with input from all authors. All authors reviewed and edited the manuscript.
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Meelker González, R., Laposchan, S., Riedel, E. et al. High-throughput chemical proteomics workflow for profiling protein citrullination dynamics. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69490-1
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DOI: https://doi.org/10.1038/s41467-026-69490-1
