Identification of mono-ADP-ribose readers using well-defined photoaffinity-based probes

Adenosine diphosphate ribosylation is a significant post-translational modification implicated in various cellular processes and diseases, yet identifying its mono-ADP-ribose readers has posed considerable challenges. Previous proteomic screenings have predominantly focused on poly-ADP-ribose, resulting in the oversight of mono-ADP-ribose readers due to undefined ADP-ribose structures with randomly placed photo-crosslinking moieties. This study introduces novel, well-defined mono-ADP-ribose photoaffinity-based probes featuring distinct diazirine and benzophenone photo-crosslinkers aimed at selectively identifying mono-ADP-ribose readers. Using human HeLa protein extracts, these probes were employed in an interactomics screening, successfully uncovering numerous known and putative mono-ADP-ribose readers, including MACROD1. This study highlights the potential of these novel probes as powerful tools for exploring the mono-ADP-ribose interactome, thereby enhancing the understanding of ADP-ribosylation signaling within cellular contexts. Proteomics data are available <i>via</i> ProteomeXchange with identifier PXD065574.