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Peer-reviewedCC BY-NC-NDNature Communications

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

  • Sara Charlotte Buch-Larsen1,2,3
  • Ivo Alexander Hendriks4,5,3
  • Kyuto Tashiro6,7
  • Jonas Damgaard Elsborg8,9,3
  • Sergey Y. Vakhrushev10,3
  • Jesper Velgaard Olsen10,8,3
  • Bernhard Lüscher11,12
  • Glen Liszczak6,13
  • Ivan Ahel9,14
  • Michael Lund Nielsen15,8,3
15 affiliations
  1. 1Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. [email protected]
  2. 2Proteomics 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. [email protected]
  3. 3University of Copenhagen
  4. 4Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. [email protected]
  5. 5Proteomics 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. [email protected]
  6. 6Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA
  7. 7University of Texas Southwestern Medical Center
  8. 8Proteomics 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
  9. 9Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
  10. 10Copenhagen Center for Glycocalyx Research, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
  11. 11Institute of Biochemistry and Molecular Biology, RWTH Aachen University, Aachen, Germany
  12. 12RWTH Aachen University
  13. 13UT Southwestern Medical Center
  14. 14University of Oxford
  15. 15Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
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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.

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Cite

BibTeXRIS
BibTeX
@article{buchlarsen2026deciphering,
  title = {{Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages}},
  author = {Sara Charlotte Buch-Larsen and Ivo Alexander Hendriks and Kyuto Tashiro and Jonas Damgaard Elsborg and Sergey Y. Vakhrushev and Jesper Velgaard Olsen and Bernhard Lüscher and Glen Liszczak and Ivan Ahel and Michael Lund Nielsen},
  journal = {Nature Communications},
  year = {2026},
  month = may,
  doi = {10.1038/s41467-026-73677-x},
  url = {https://doi.org/10.1038/s41467-026-73677-x},
  note = {CC BY-NC-ND},
}
RIS
TY  - JOUR
TI  - Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages
AU  - Sara Charlotte Buch-Larsen
AU  - Ivo Alexander Hendriks
AU  - Kyuto Tashiro
AU  - Jonas Damgaard Elsborg
AU  - Sergey Y. Vakhrushev
AU  - Jesper Velgaard Olsen
AU  - Bernhard Lüscher
AU  - Glen Liszczak
AU  - Ivan Ahel
AU  - Michael Lund Nielsen
PY  - 2026
DA  - 2026/05/27
JO  - Nature Communications
DO  - 10.1038/s41467-026-73677-x
UR  - https://doi.org/10.1038/s41467-026-73677-x
AB  - 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.
SN  - 2041-1723
ER  -