A Large-Scale Method to Measure the Stoichiometries of Protein Poly-ADP-Ribosylation

Poly-ADP-ribosylation (PARylation) is a reversible post-translational modification that occurs in higher eukaryotes. While thousands of PARylated substrates have been identified, the specific biological functions of most PARylated proteins remain elusive. PARylation stoichiometry is a critical parameter to assess the potential functions of a PARylated protein. Here, we developed a large-scale strategy to measure the stoichiometries of protein PARylation. By integrating chemically mild cell lysis conditions, boronate enrichment, and carefully designed titration experiments, we were able to determine the PARylation stoichiometries for a total of 235 proteins. Importantly, this approach enables the capture of all PARylation events, regardless of their amino acid acceptor linkages. We revealed that PARylation occupancy spans over 3 orders of magnitude. However, most PARylation events occur at low stoichiometric values (median 0.58%). Notably, we observed that high-stoichiometry PARylation (>1%) predominantly targets proteins involved in transcription regulation and chromatin remodeling. Thus, our study provides a system-scale, quantitative view of PARylation stoichiometries under genotoxic conditions, which serves as an invaluable resource for future functional studies of this important protein post-translational modification.