Cell‐Permeable Nicotinamide Adenine Dinucleotides for Exploration of Cellular Protein ADP‐Ribosylation

Abstract Posttranslational modifications (PTMs) greatly enhance the functional diversity of proteins, surpassing the number of gene‐encoded variations. One intriguing PTM is ADP‐ribosylation, which utilizes nicotinamide adenine dinucleotide (NAD + ) as a substrate and is essential in cell signaling pathways regulating cellular responses. Here, we report the first cell‐permeable NAD + analogs and demonstrate their utility for investigating cellular ADP‐ribosylation. Using a desthiobiotin‐labelled analog for affinity enrichment of proteins that are ADP‐ribosylated in living cells under oxidative stress, we identified protein targets associated with host‐virus interactions, DNA damage and repair, protein biosynthesis, and ribosome biogenesis. Most of these targets have been noted in various literature sources, highlighting the potential of our probes for cellular ADP‐ribosylome studies.