ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. To facilitate live-cell profiling of ADP-ribosylated proteins, we show that 2-alkyne-adenosine (2YnAd) is

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... ically incorporated in mammalian cells and enables fluorescent detection and robust affinity enrichment of the modified proteins. We then present an integrated chemical biology approach that involves simultaneous metabolic incorporation of 2YnAd and the previously reported 6-alkyne-adenosine (6YnAd) in live cells followed by click chemistry with a capture reagent to facilitate highly sensitive and comprehensive enrichment of the modified proteins. By combining this dual metabolic labelling strategy with the tandem mass tag (TMT) isobaric mass spectrometry, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib. Our study provides insight into the wider scope of PARP targets and will help to advance further characterisation of their functional roles.