Abstract
Poly(ADP-ribosyl)ation (PARylation), i.e., the formation of the nucleic acid-like biopolymer poly(ADP-ribose) (PAR), is an essential posttranslational modification carried out by poly(ADP-ribose) polymerases (PARPs). While PAR levels are low under physiological conditions, they can transiently increase more than 100-fold upon induction of genotoxic stress. The accurate quantitation of cellular PAR with high sensitivity is of critical importance to understand the role of PARylation in cellular physiology and pathophysiology and to determine the pharmacodynamic efficiencies of clinically relevant PARP inhibitors, which represent a novel class of promising chemotherapeutics. Previously, we have developed a bioanalytical platform based on isotope dilution mass spectrometry (LC-MS/MS) to quantify cellular PAR with unequivocal chemical specificity in absolute terms with femtomol sensitivity (Martello et al. ACS Chem Biol 8(7):1567–1575, 2013). This method enables the analysis of steady-state levels, as well as stress-induced levels of PAR in various biological systems including cell lines, mouse tissues, and primary human lymphocytes. It has a wide range of potential applications in basic research, as well as in drug development (Martello et al. ACS Chem Biol 8(7):1567–1575, 2013; Mangerich et al. Toxicol Lett 244:56–71, 2016). Here, we present an improved and adjusted version of the original protocol by Martello/Mangerich et al., which uses UPLC-MS/MS instrumentation.
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Acknowledgments
This work was supported by the DFG-funded Konstanz Research School Chemical Biology (KoRSCB), by the Collaborative Research Center (CRC) 969, and by the German Federal Ministry of Defense.
This chapter represents an updated protocol for the quantitation of poly(ADP-ribose) by isotope dilution mass spectrometry in cells and tissues, which is based on a previous publication by Martello/Mangerich et al. [1]. Parts of the original publication are reprinted with permission of the American Chemical Society (ACS).
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Zubel, T., Martello, R., Bürkle, A., Mangerich, A. (2017). Quantitation of Poly(ADP-Ribose) by Isotope Dilution Mass Spectrometry. In: Tulin, A. (eds) Poly(ADP-Ribose) Polymerase. Methods in Molecular Biology, vol 1608. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6993-7_1
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DOI: https://doi.org/10.1007/978-1-4939-6993-7_1
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