New proteomics strategy reveals hidden ADP-ribosylation signaling networks

ADP-ribosylation is a protein modification that regulates a wide range of cellular processes, including DNA repair, stress responses, metabolism, and immune signaling. Despite being discovered more than 60 years ago, fundamental questions remain unanswered about where and how this modification occurs inside cells.

One reason is technical. Certain forms of ADP-ribosylation are highly unstable and can disappear during standard laboratory workflows before they can be detected by mass spectrometry.

In a new study published in Nature Communications, researchers from the Nielsen Laboratory at the Department of Molecular Biology and Genetics, Aarhus University, together with collaborators from the University of Oxford, University of Texas Southwestern Medical Center, RWTH Aachen University, and the University of Copenhagen, have addressed this challenge by refining one of the most widely used proteomics approaches for studying ADP-ribosylation.

A long-standing challenge in the field

Over the past decade, mass spectrometry-based proteomics has transformed the study of ADP-ribosylation and enabled the identification of thousands of modification sites across the proteome. However, most studies have focused on relatively stable forms of the modification.

Other researchers have previously shown that ADP-ribosylation attached to glutamate and aspartate residues is particularly sensitive to standard sample preparation conditions. As a result, these modifications have remained difficult to study systematically and have likely been underrepresented in existing datasets.

“ADP-ribosylation is a highly dynamic modification, and some forms are surprisingly fragile,” says Professor Michael L. Nielsen, senior author of the study. “We wanted to understand why these modifications were being lost and whether we could adapt our existing proteomics platform to preserve them.”

The team systematically investigated how different sample preparation conditions affect ADP-ribosylation stability and identified key steps that lead to the loss of glutamate- and aspartate-linked modifications.

Expanding the capabilities of a widely used proteomics platform

The researchers then refined their Af1521-based enrichment workflow, a strategy that has become a cornerstone technology for large-scale ADP-ribosylation analysis.

By combining acidic sample preparation conditions with an engineered version of the Af1521 enrichment reagent, the researchers were able to preserve and identify labile ADP-ribosylation events that were previously difficult to detect using this workflow.

“This work shows that the Af1521 platform can capture a much broader spectrum of ADP-ribosylation biology than previously appreciated,” says Michael L. Nielsen. “It allows us to investigate modification events that have largely remained hidden in conventional analyses.”

Revealing cytokine-driven signaling networks

To demonstrate the biological potential of the approach, the researchers applied the workflow to study cytokine signaling pathways.

The analyses uncovered hundreds of glutamate- and aspartate-linked ADP-ribosylation sites across cellular proteins involved in immune regulation and antiviral responses. The study revealed that these modifications preferentially target cytoplasmic signaling networks and display distinct patterns depending on cell type and cytokine stimulation.

The researchers also identified extensive ADP-ribosylation associated with interferon signaling pathways and uncovered evidence linking ADP-ribosylation to ubiquitin-dependent regulatory processes.

Together, the results provide one of the most comprehensive resources to date for studying glutamate- and aspartate-linked ADP-ribosylation and offer new insights into how cells respond to inflammatory and antiviral signals.

Opening new opportunities for ADP-ribosylation research

The researchers believe the study will help accelerate efforts to understand the biological functions of different ADP-ribosylation linkage types and their roles in health and disease.

“For many years, researchers have debated which amino acids are modified by ADP-ribosylation under different biological conditions,” says Michael L. Nielsen. “By improving our ability to observe these elusive modifications directly in cells, we can begin to answer some of these fundamental questions and gain a more complete picture of ADP-ribosylation signaling.”

The study was published in Nature Communications under the title ‘Deciphering cytokine-driven ADP-ribosylation signaling networks via Af1521-based mass spectrometry analysis of labile Glu/Asp-linkages’.