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Poly(ADP-Ribose) Polymerase pp 137–148Cite as

Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography–Tandem Mass Spectrometry

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1608))

Abstract

Protein ADP-ribosylation is a covalent, reversible posttranslational modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs). Proteins can be either mono- or poly-ADP-ribosylated under a variety of physiological and pathological conditions. To understand the functional contribution of protein ADP-ribosylation to normal and disease/stress states, modified protein and corresponding ADP-ribose acceptor site identification is crucial. Since ADP-ribosylation is a transient and relatively low abundant PTM, systematic and accurate identification of ADP-ribose acceptor sites has only recently become feasible. This is due to the development of specific ADP-ribosylated protein/peptide enrichment methodologies, as well as technical advances in high-accuracy liquid chromatography–tandem mass spectrometry (LC-MS/MS). The standardized protocol described here allows the identification of ADP-ribose acceptor sites in in vitro ADP-ribosylated proteins and will, thus, contribute to the functional characterization of this important PTM.

*These authors contributed equally to this chapter.

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Acknowledgments

The authors would like to thank Paolo Nanni (member of the Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Zurich, Switzerland) for advice and technical assistance. We also thank Felix R. Althaus (Institute of Pharmacology and Toxicology, University of Zurich-Vetsuisse) for providing hPARG-expressing baculovirus. Stephan Christen and Deena Leslie Pedrioli (both University of Zurich) provided editorial assistance and critical input during the writing. Work on ADP-ribosyltransferases in the laboratory of M.O.H is supported by Kanton of Zurich and the Swiss National Science Foundation (310030_157019).

Mario Leutert and Vera Bilan contributed equally to this chapter.

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    Authors and Affiliations

    1. Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

      Mario Leutert, Vera Bilan & Michael O. Hottiger

    2. Molecular Life Science PhD Program of the Life Science Zurich Graduate School, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

      Mario Leutert & Vera Bilan

    3. Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

      Peter Gehrig

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    1. Mario Leutert
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    2. Vera Bilan
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    3. Peter Gehrig
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    4. Michael O. Hottiger
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    Correspondence to Michael O. Hottiger .

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    Editors and Affiliations

    1. Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA

      Alexei V. Tulin

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    Leutert, M., Bilan, V., Gehrig, P., Hottiger, M.O. (2017). Identification of ADP-Ribose Acceptor Sites on In Vitro Modified Proteins by Liquid Chromatography–Tandem 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_10

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    • DOI: https://doi.org/10.1007/978-1-4939-6993-7_10

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6992-0

    • Online ISBN: 978-1-4939-6993-7

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