Dataset Information

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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Ivo Hendriks

LAB HEAD: Sara Buch-Larsen

PROVIDER: PXD075810 | Pride | 2026-05-27

REPOSITORIES: Pride

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			Action	DRS
	20260119_AST3_NEO6_SCL-IAH_50ID_ADPr-TP_A549_Control_01.raw	Raw
	20260119_AST3_NEO6_SCL-IAH_50ID_ADPr-TP_A549_Control_02.raw	Raw
	20260119_AST3_NEO6_SCL-IAH_50ID_ADPr-TP_A549_Control_03.raw	Raw
	20260119_AST3_NEO6_SCL-IAH_50ID_ADPr-TP_A549_Control_04.raw	Raw
	20260119_AST3_NEO6_SCL-IAH_50ID_ADPr-TP_A549_Control_x01.raw	Raw

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Dataset Information

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

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