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.
Highlights
Protein ADP-ribosylation is a post-translational modification (PTM) where members of a family of enzymes known as ADP-ribosyltransferases covalently link ADP-ribose moieties derived from NAD+ to the side chains of several possible amino acid residues on target proteins
It has been demonstrated that 6-alkyne adenosine (6YnAd), a compound that was previously demonstrated to be suitable for labelling poly(A) tails of mRNAs in mammalian cells[9], enables sensitive fluorescence profiling of ADP-ribosylated proteins in live cells[10]
Functionalised full-length NAD+ analogues have recently emerged as promising tools for fluorescence-based visualisation of ADP-ribosylation in cell-lysates[11,16,17,18] and in cells[19,20]
Summary
Protein ADP-ribosylation is a post-translational modification (PTM) where members of a family of enzymes known as ADP-ribosyltransferases (or PARPs) covalently link ADP-ribose moieties derived from NAD+ to the side chains of several possible amino acid residues (glutamate, aspartate, arginine, lysine, cysteine, serine) on target proteins. Following affinity capture of the labelled proteins on NeutrAvidin-Agarose resin, the combination of in-gel fluorescence imaging (Fig. 1C) and Western blot analysis using an anti-pan-ADP-ribose antibody (Fig. 1D)[11] confirmed significant enrichment of ADP-ribosylated proteins in both 2YnAd and 6YnAd treated cells relative to the input control lanes
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