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Methods Mol Biol. 2017;1608:137-148. doi: 10.1007/978-1-4939-6993-7_10.

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

Author information

1
Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
2
Molecular Life Science PhD Program of the Life Science Zurich Graduate School, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
3
Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
4
Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. michael.hottiger@dmmd.uzh.ch.

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.

KEYWORDS:

ADP-ribosylation; ADP-ribosylome; ARTD; Mass spectrometry; PARG; PARP; Phosphoenrichment; Ti4+-IMAC enrichment

PMID:
28695508
DOI:
10.1007/978-1-4939-6993-7_10
[Indexed for MEDLINE]

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