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

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.