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Methods Mol Biol. 2017;1608:149-162. doi: 10.1007/978-1-4939-6993-7_11.

Proteome-Wide Identification of In Vivo ADP-Ribose Acceptor Sites by Liquid Chromatography-Tandem Mass Spectrometry.

Author information

1
Department of Proteomics, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, 2200, Copenhagen, Denmark.
2
Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
3
Molecular Life Science PhD Program of the Life Science Zurich Graduate School, University of Zurich/ETH Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
4
Department of Proteomics, Faculty of Health and Medical Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, 2200, Copenhagen, Denmark. michael.lund.nielsen@cpr.ku.dk.

Abstract

ADP-ribosylation is a posttranslational modification (PTM) that affects a variety of cellular processes. In recent years, mass spectrometry (MS)-based proteomics has become a valuable tool for studying ADP-ribosylation. However, studying this PTM in vivo in an unbiased and sensitive manner has remained a difficult challenge. Here, we describe a detailed protocol for unbiased analysis of ADP-ribosylated proteins and their ADP-ribose acceptor sites under physiological conditions. The method relies on the enrichment of mono-ADP-ribosylated peptides using the macrodomain Af1521 in combination with liquid chromatography-high-resolution tandem MS (LC-MS/MS). The 5-day protocol explains the step-by-step enrichment and identification of ADP-ribosylated peptides from cell culture stage all the way through to data processing using the MaxQuant software suite.

KEYWORDS:

ADP-ribosylation; ADP-ribosylome; Af1521 macrodomain enrichment; Affinity purification; Mass spectrometry; PARG; Proteomics

PMID:
28695509
DOI:
10.1007/978-1-4939-6993-7_11
[Indexed for MEDLINE]

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