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Mol Cell Proteomics. 2017 May;16(5):949-958. doi: 10.1074/mcp.O116.065623. Epub 2017 Mar 21.

New Quantitative Mass Spectrometry Approaches Reveal Different ADP-ribosylation Phases Dependent On the Levels of Oxidative Stress.

Author information

1
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
2
§Molecular Life Science (MLS) program of the Life Science Zurich Graduate School, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
3
¶Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
4
‖Leiden Institute of Chemistry, Department of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
5
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; michael.hottiger@dmmd.uzh.ch.

Abstract

Oxidative stress is a potent inducer of protein ADP-ribosylation. Although individual oxidative stress-induced ADP-ribosylated proteins have been identified, it is so far not clear to which extent different degrees of stress severity quantitatively and qualitatively alter ADP-ribosylation. Here, we investigated both quantitative and qualitative changes of the hydrogen peroxide (H2O2)-induced ADP-ribosylome using a label-free shotgun quantification and a parallel reaction monitoring (PRM) mass spectrometry approach for a selected number of identified ADP-ribosylated peptides. Although the major part of the basal HeLa ADP-ribosylome remained unchanged upon all tested H2O2 concentrations, some selected peptides change the extent of ADP-ribosylation depending on the degree of the applied oxidative stress. Low oxidative stress (i.e. 4 μm and 16 μm H2O2) caused a reduction in ADP-ribosylation of modified proteins detected under untreated conditions. In contrast, mid to strong oxidative stress (62 μm to 1 mm H2O2) induced a significant increase in ADP-ribosylation of oxidative stress-targeted proteins. The application of the PRM approach to SKOV3 and A2780, ovarian cancer cells displaying different sensitivities to PARP inhibitors, revealed that the basal and the H2O2-induced ADP-ribosylomes of SKOV3 and A2780 differed significantly and that the sensitivity to PARP inhibitors correlated with the level of ARTD1 expression in these cells. Overall, this new PRM-MS approach has proven to be sensitive in monitoring alterations of the ADP-ribosylome and has revealed unexpected alterations in proteins ADP-ribosylation depending on the degree of oxidative stress.

PMID:
28325851
PMCID:
PMC5417832
DOI:
10.1074/mcp.O116.065623
[Indexed for MEDLINE]
Free PMC Article

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