Proteomic Dataset of Plant ADP-Ribosylated Proteins Identified via Click Chemistry-Based Chemoproteomic Strategy

ADP-ribosylation is an important and reversible post-translational modification in eukaryotes. In plants, accumulating evidence suggests that it contributes to immunity, stress responses, and genome maintenance, yet systematic characterization of plant ADP-ribosylated proteins remains technically challenging. Previous studies have identified plant ADP-ribosylation-associated proteins through protein microarray-based screening, targeted biochemical analyses, and mass spectrometric validation. For example, an Arabidopsis protein microarray coupled with in vitro PARylation assays enabled global identification of candidate PARylation targets, and subsequent studies established biologically relevant ADP-ribosylation events on GRP7 and RIN4/NOI proteins, DAWDLE, and SZF1/SZF2. Despite these advances, a broadly applicable and experimentally tractable workflow for systematic analysis of ADP-ribosylated proteins from plant samples is still lacking, limiting comprehensive exploration of the plant ADP-ribosylome and its underlying mechanisms. To address this limitation, we established a click chemistry-based chemoproteomic workflow for identifying plant ADP-ribosylated proteins. This strategy was adapted from a metabolic labeling approach previously developed in mammalian cells, in which the alkyne-functionalized adenosine analogue N6-propargyl adenosine (N6pA) (Supplemental Figure 1A) is incorporated into ADP-ribosylated proteins in vivo and subsequently conjugated to reporter tags through copper-catalyzed azide-alkyne cycloaddition. In our workflow, plant seedlings were metabolically labeled with N6pA, followed by cell lysis and CuAAC-mediated conjugation to biotin-PEG3-azide. The labeled proteins were then enriched with streptavidin beads and analyzed either by immunoblotting or by tryptic digestion coupled with LC-MS/MS, enabling proteome-scale detection of ADP-ribosylated proteins (Figure 1A). By adapting this chemoproteomic strategy to plant samples, we established a robust platform for the enrichment and systematic identification of plant ADP-ribosylated proteins.