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Protein citrullination, the conversion of arginine to citrulline by peptidylarginine deiminases (PADs), plays crucial roles in both normal physiology and disease states including rheumatoid arthritis, neurodegenerative disorders, and cancer. Despite its biological significance, comprehensive analysis of citrullination has been hampered by technical challenges including the small mass shift (0.98 Da), low abundance, and lack of effective enrichment methods.

To address these limitations, González et al. developed a high-throughput chemical workflow for global citrullinome profiling combining glyoxal-based derivatisation with a clickable-cleavable linker strategy. The workflow utilises commercially available reagents and is compatible with 96-well plate processing, enabling scalable analysis of complex samples.

For mass spectrometry analysis, the team from the Lee lab employed an Orbitrap Fusion Lumos for synthetic peptides and mouse brain samples. Neutrophil samples were analysed using an Orbitrap Astral coupled to a Vanquish Neo with a Nanospray Flex ion source, peptides being separated on an IonOpticks Aurora Rapid 8×75 XT C18 UHPLC column.

The optimised workflow yielded 4- to 10-fold increases in peptide identifications and 53- to 155-fold increases in citrullinated peptide intensity. In activated neutrophils, the researchers identified up to 1,700 citrullinated peptides on 580 proteins, revealing extensive modification of histones and structural proteins during NET formation.

This methodology significantly expands our understanding of citrullination’s role beyond histone H3, identifying widespread modification of linker histones and nuclear envelope proteins that may contribute to chromatin decondensation and nuclear rupture during NET formation.

The workflow provides a valuable resource for investigating citrullination’s role in both physiological processes and disease pathogenesis.

Publication
bioRxiv

Authors

Rebecca Meelker González, Sophia Laposchan, Erik Riedel, Anna Fürst, Naomi O’Sullivan, Wassim Gabriel, Mathias Wilhelm, Percy A. Knolle, Guillaume Médard, Bernhard Kuster, & Chien-Yun Lee;

Title

High-throughput chemical proteomics workflow for profiling protein citrullination dynamics