Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages

Journal article

ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues, but glutamate/aspartate-linked ADPr (Glu/Asp-ADPr) is labile and remains challenging to detect using conventional mass spectrometry (MS)-based workflows. Using synthetic peptides, we show that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, and by hydrolysis via wildtype Af1521. We developed an acidic enrichment workflow incorporating an Af1521 mutant that preserves Glu/Asp-ADPr, enabling site-specific, system-wide MS analysis. In cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp- and >200 Cys-ADPr sites. Glu/Asp-ADPr marks cytoplasmic, immune-related protein networks, contrasting with nuclear Ser-ADPr. Quantitative profiling revealed reproducible, cell type- and treatment-specific patterns. PARP10-mediated Glu/Asp ADPr of ubiquitin indicates direct crosstalk with ubiquitin signaling pathways. Interferon treatments revealed conserved antiviral PARP networks extensively modified on Glu/Asp residues. Together, our work establishes a robust MS-based workflow and provides a resource of site-specific ADPr events, revealing residue-specific ADPr in innate immune signaling.

Authors: Buch-Larsen, SC; Hendriks, IA; Tashiro, K; Elsborg, JD; Vakhrushev, SY

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Nature Research
• Journal: Nature Communications
• Publication date: 2025-11-04
• DOI: 10.1038/s41467-026-73677-x
• EISSN: 2041-1723
• ISSN: 2041-1723
• Language: English
• Keywords: Immune system; HEK 293 cells; Proteomics; Quantitative proteomics; Regulator; Phosphoproteomics; Immunoprecipitation; Ubiquitin; Multiplex