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

TL;DR

This paper introduces a new method to measure how often proteins are modified by PARylation, revealing that most modifications occur at low levels and are linked to specific biological functions.

Contribution

A novel large-scale method to quantify PARylation stoichiometry across hundreds of proteins, capturing all amino acid acceptor linkages.

Findings

PARylation occupancy varies over three orders of magnitude, with most events occurring at low stoichiometry (median 0.58%).

High-stoichiometry PARylation primarily targets proteins involved in transcription regulation and chromatin remodeling.

The method enables comprehensive quantification of PARylation under genotoxic conditions.

Abstract

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.

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