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

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: PXD070310 | Pride | 2026-05-27

REPOSITORIES: Pride

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			Action	DRS
	20220915_LUMOS1_LC6_SCL-IAH_ADPr-mix-pep_0h_02.raw	Raw
	20220915_LUMOS1_LC6_SCL-IAH_ADPr-mix-pep_0h_03.raw	Raw
	20220915_LUMOS1_LC6_SCL-IAH_ADPr-mix-pep_0h_04.raw	Raw
	20220915_LUMOS1_LC6_SCL-IAH_ADPr-mix-pep_0h_05.raw	Raw
	20220915_LUMOS1_LC6_SCL-IAH_ADPr-mix-pep_0h_06.raw	Raw

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

Project description: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.

2026-05-27 | PXD075810 | Pride

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome (part 2)

Project description:Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADPribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.

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An HPF1/PARP1-based chemical biology strategy for exploring ADP-ribosylation

Project description:Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive PTM in essential cellular processes. Here we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely-modified peptides. Integrating this streamlined methodology with phage display technology, we have developed the first site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. By enabling mono-ADPr proteomics and poly-to-mono comparisons at the modification site level, we have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology/recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.

2020-11-17 | PXD020589 | Pride

A site-specific analysis of the ADP-Ribosylome unveils Homogeneous Serine ADP-Ribosylation across wild-type and BRCA-mutant Breast Cancer cell lines

Project description:ADP-ribosylation (ADPr) signaling plays a crucial role in the DNA damage response. Inhibitors against the main enzyme catalyzing ADPr after DNA damage – PARP1 – are used as targeted therapies against breast cancers with BRCA1/2 mutations. However, development of resistance to PARP inhibitors (PARPi) is a major obstacle in treating patients. To better understand the role of ADPr in PARPi sensitivity, we used Liquid Chromatography-Mass Spectrometry (LC-MS) for systems level analysis of the ADP-ribosylome in six breast cancer cell lines with different PARPi sensitivities. We identified 1632 sites on 777 proteins, primarily on serine residues, with a high site overlap of DNA damage-related proteins across all cell lines, demonstrating high conservation in ADPr signaling after DNA damage. We furthermore observed site-specific differences in ADPr intensities in PARPi-sensitive BRCA mutants, and unique ADPr sites in PARPi-resistant BRCA mutant cells, which we notably show to have low PARG levels and longer PARP1 ADPr chains.

2024-08-02 | PXD047613 | Pride

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signaling

Project description:In the mammalian DNA damage response, ADP-ribosylation signaling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognizes damaged DNA and catalyzes the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signaling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the Parp1:Hpf1 complex. Moreover, our structural and biochemical data reveal a new mechanism of mono-Ser-ADPr removal by Drosophila Parg.

2023-05-26 | PXD036512 | Pride

A combined gas phase separation strategy for ADP-ribosylated peptides

Project description:ADP-ribosylation (ADPr) is a posttranslational modification that is best studied using mass spectrometry. Method developments that are permissive to low inputs or baseline levels of protein ribosylation represent the next frontier in the field. High-field asymmetric waveform ion mobility spectrometry (FAIMS) reduces peptide complexity in gas phase, providing a means to achieve maximal ADPr peptide sequencing depth. We therefore investigated the extent to which FAIMS with or without traditional gas-phase fractionation/separation (GPS) can increase the number of ADPr peptides. We examined ADPr peptides enriched from mouse spleens. We gleaned additional insight by also reporting findings from the corresponding non-ADPr peptide contaminants, and the peptide inputs for ADPr peptide enrichment. At increasingly higher compensation voltages, ADPr peptides were more stable whereas the non-ADPr peptides were filtered out. A combination of 3 GPS survey scans, each with 8 compensation voltages resulted in 790 high-confidence ADPr peptides, compared to 90 with GPS alone. A simplified acquisition strategy requiring only two injections corresponding to two MS1 scan ranges coupled to optimized compensation voltage settings, provided 402 ADPr peptides corresponding to 234 ADPr proteins. We conclude that our combined gas phase separation strategy is a valuable addition to any ADP-ribosylome workflow.

2023-08-30 | PXD040898 | Pride

A Novel Spectral Annotation Strategy Streamlines Reporting of mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-gamma

Project description:Mass spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4-hour exposure to systemic IFN-gamma. HCD collision energy experiments were first performed on the Obitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization.

2021-11-02 | PXD027454 | Pride

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site

Project description:Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-site identification and localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1 dependent in vivo ADPr-acceptor. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

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Project description:ADP-ribosylation is a posttranslational modification whose HCD products are dominated by complete or partial modification losses, complicating peptide sequencing and acceptor site localization efforts. We tested whether in-source CID performed on a quadrupole Orbitrap could convert ADPr to the smaller phosphoribose-H2O derivative to facilitate HCD-dependent peptide sequencing. Human macrophage like cell line THP-1-derived ADP-ribosyl (ADPr) peptides were analyzed on a quadrupole Orbitrap. We monitored the interconversion of ADPr (+541.061 Da) to phosphoribosyl-H2O (+193.997 Da) peptides while varying the source and high-field asymmetric waveform ion mobility mass spectrometry (FAIMS) compensation voltages. Xcorr and ptmRS were used to evaluate peptide sequencing and acceptor site confidence, respectively. In-source CID-HCD-derived phosphoribosyl-H2O acceptor sites were compared to those determined by EThcD, performed on a quadrupole ion trap Orbitrap. Interconversion of ADPr peptides to their phosphoribosyl-H2O derivatives increased with increasing source voltage (up to 50V), as judged by monitoring the corresponding modification loss ([adenosine monophosphate/AMP]+) and the number of identified phosphoribosyl-H2O peptide identifications. The average Xcorr increased from 1.36 (ADPr) to 2.26 (phosphoribosyl-H2O), similar to that achieved with EThcD for ADPr peptides (2.29). The number of high-confidence acceptor sites (>95%) also increased, from 31% (ADPr) to 70% (phosphoribosyl-H2O), which was comparable to EThcD (70%). In-source CID converts ADP-ribosyl to phosphoribosyl-H2O peptides that are more amenable to HCD-dependent peptide sequencing, providing an alternative method for acceptor site determination when ETD-based methods are not available.

2024-12-05 | PXD055586 | Pride

Serine ADP-ribosylation depends on HPF1

Project description:We report that serine ADPr is strictly dependent on HPF1 (histone PARylation factor 1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. We identified three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on hundreds of other targets indicates that serine ADPr is a widespread modification.

2017-02-10 | PXD005627 | Pride

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