{ "abstract": "ADP-ribosylation (ADPr) is a regulatory post-translational modification targeting nine amino acid residues. Glutamate- and aspartate-linked ADPr (Glu/Asp-ADPr) is chemically unstable during sample preparation for conventional mass spectrometry (MS)-based proteomics workflows, limiting its detection. Here, we systematically assessed the stability of ADPr linkages using synthetic peptides and confirmed that ester-linked Glu/Asp-ADPr is lost under alkaline conditions, elevated temperatures, or through hydrolysis by wildtype Af1521. We established an acidic enrichment workflow encompassing an Af1521 mutant, robustly preserving Glu/Asp-ADPr enabling their site-specific and systems-wide MS analysis. Applying this strategy to cytokine-stimulated A549 and HeLa cells, we identified >600 Glu/Asp-ADPr and >200 Cys-ADPr sites. Our analysis uncovered that Glu/Asp-ADPr marks distinct cytoplasmic protein networks enriched in immune functions, contrasting with Ser-ADPr typically observed on nuclear and chromatin-associated proteins. Quantitative profiling revealed reproducible ADPr patterns specific to cell type and treatment. Notably, PARP10 promoted Glu/Asp modification of ubiquitin, highlighting crosstalk between ADPr and ubiquitin signaling. Across interferon treatments, we identified a conserved network of antiviral PARPs and associated cofactors extensively modified on Glu/Asp residues, emphasizing residue-specific ADPr as a regulator of innate immune signaling. Together, our work establishes an MS-based proteomics workflow for identification of Glu/Asp-ADPr, provides a resource of site-specific modification events, and reveals residue-specific ADPr dynamics in immune signaling.", "abstract_shown": true, "affiliations": [ "Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. sara.larsen@cpr.ku.dk", "Proteomics program, Novo Nordisk Foundation Center for Protein Research, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. sara.larsen@cpr.ku.dk", "University of Copenhagen", "Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. ivo.hendriks@cpr.ku.dk", "Proteomics program, Novo Nordisk Foundation Center for Protein Research, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. ivo.hendriks@cpr.ku.dk", "Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA", "University of Texas Southwestern Medical Center", "Proteomics program, Novo Nordisk Foundation Center for Protein Research, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark", "Sir William Dunn School of Pathology, University of Oxford, Oxford, UK", "Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark", "Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Aachen, Germany", "RWTH Aachen University", "UT Southwestern Medical Center", "University of Oxford", "Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark" ], "article_type": null, "arxiv": null, "author_total": 10, "authors": [ { "affs": [ 0, 1, 2 ], "name": "Sara Charlotte Buch-Larsen", "orcid": "0000-0001-6250-5467" }, { "affs": [ 3, 4, 2 ], "name": "Ivo Alexander Hendriks", "orcid": "0000-0002-1439-3701" }, { "affs": [ 5, 6 ], "name": "Kyuto Tashiro", "orcid": "0000-0002-0174-1789" }, { "affs": [ 7, 8, 2 ], "name": "Jonas Damgaard Elsborg", "orcid": "0000-0002-1506-4280" }, { "affs": [ 9, 2 ], "name": "Sergey Y. Vakhrushev", "orcid": "0000-0002-0418-5765" }, { "affs": [ 9, 7, 2 ], "name": "Jesper Velgaard Olsen", "orcid": "0000-0002-4747-4938" }, { "affs": [ 10, 11 ], "name": "Bernhard Lüscher", "orcid": "0000-0002-9622-8709" }, { "affs": [ 5, 12 ], "name": "Glen Liszczak", "orcid": "0000-0001-8194-5281" }, { "affs": [ 8, 13 ], "name": "Ivan Ahel", "orcid": "0000-0002-9446-3756" }, { "affs": [ 14, 7, 2 ], "name": "Michael Lund Nielsen", "orcid": "0000-0002-0067-9039" } ], "bibtex": "https://research.draconitic.com/paper/bxdk297q4zx22.bib", "cited_by": 9, "doi": "10.1038/s41467-026-73677-x", "fulltext_url": null, "id": "bxdk297q4zx22", "issns": [ "2041-1723" ], "landing_url": "https://doi.org/10.1038/s41467-026-73677-x", "language": "en", "licence": "CC-BY-NC-ND", "licence_label": "CC BY-NC-ND", "licence_source": "journal", "licence_url": "https://creativecommons.org/licenses/by-nc-nd/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "1607|Advanced Proteomics Techniques and Applications" ] ], "openalex": "W4415887598", "pdf_url": "https://www.nature.com/articles/s41467-026-73677-x_reference.pdf", "pmcid": "PMC13388985", "pmid": "42203779", "published": "2026-05-27", "publisher": "Nature Portfolio", "ris": "https://research.draconitic.com/paper/bxdk297q4zx22.ris", "sources": [ { "first_seen": "2026-10-30T13:26:44Z", "native_id": "W4415887598", "source": "openalex", "url": "https://doi.org/10.1038/s41467-026-73677-x" } ], "subjects": [ { "field": "chemistry", "origin": "openalex:1607|Advanced Proteomics Techniques and Applications", "subfield": "physical" } ], "title": "Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages", "title_text": "Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages", "type": "peer-reviewed", "updated_at": "2026-11-09T13:21:03Z", "url": "https://research.draconitic.com/paper/bxdk297q4zx22", "venue": "Nature Communications", "versions": [] }