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Sci Rep. 2019 Apr 30;9(1):6655. doi: 10.1038/s41598-019-43154-1.

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation.

Author information

1
Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
2
Department of Chemistry, Durham University, Stockton Road, Durham, DH1 3LE, UK.
3
Institute of Functional Epigenetics, Helmholtz Zentrum München, Neuherberg, Germany.
4
Mass Spectrometry Proteomics Platform, The Francis Crick Institute, London, NW1 1AT, UK.
5
Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, 21205, USA.
6
Faculty of Medicine, National Heart & Lung Institute, Vascular Science Section, Hammersmith Campus, London, UK.
7
Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK. p.dimaggio@imperial.ac.uk.

Abstract

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. We herein show that an adenosine analogue with a terminal alkyne functionality at position 2 of the adenine (2-alkyne adenosine or 2YnAd) is suitable for selective enrichment, fluorescence detection and mass spectrometry proteomics analysis of the candidate ADP-ribosylome in mammalian cells. Although similar labelling profiles were observed via fluorescence imaging for 2YnAd and 6YnAd, a previously reported clickable NAD+ precursor, quantitative mass spectrometry analysis of the two probes in MDA-MB-231 breast cancer cells revealed a significant increase in protein coverage of the 2YnAd probe. To facilitate global enrichment of ADP-ribosylated proteins, we developed a dual metabolic labelling approach that involves simultaneous treatment of live cells with both 2YnAd and 6YnAd. By combining this dual metabolic labelling strategy with highly sensitive tandem mass tag (TMT) isobaric mass spectrometry and hierarchical Bayesian analysis, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib.

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