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

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.