biostudies-literatureUnknownvan der Heijden FLAMDutch Research Council (NWO)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-RSC chemical biologyhttps://www.ebi.ac.uk/biostudies/studies/S-EPMC12679546biostudies-literatureIdentification of mono-ADP-ribose readers using well-defined photoaffinity-based probes.PMC12679546OCENW.KLEIN.497van der Heijden FLAMKondyli SFilippov DVBleijerveld OVermeulen MWeijers SAfalseIdentification 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-2025-01-01T00:00:00Z2025 Nov2026-06-05T23:35:11.783Z2026-05-23T03:13:54.142ZS-EPMC126795464135819610.1039/d5cb00176e