New Quantitative Mass Spectrometry Approaches Reveal Different ADP-ribosylation Phases Dependent On the Levels of Oxidative Stress.
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 H2O2concentrations, 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.
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.|§Molecular Life Science (MLS) program of the Life Science Zurich Graduate School, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Vera
V
Bilan
¶Functional Genomics Center Zurich, University of Zurich/ETH Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Nathalie
N
Selevsek
‖Leiden Institute of Chemistry, Department of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Hans A V
HAV
Kistemaker
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.|§Molecular Life Science (MLS) program of the Life Science Zurich Graduate School, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Jeannette
J
Abplanalp
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
Roxane
R
Feurer
‖Leiden Institute of Chemistry, Department of Bio-organic Synthesis, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Dmitri V
DV
Filippov
From the ‡Department of Molecular Mechanisms of Disease, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland; michael.hottiger@dmmd.uzh.ch.
Michael O
MO
Hottiger
D000074744:ADP-Ribosylation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D018384:Oxidative Stress / Q000187:drug effects; D010455:Peptides / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism
D010455:Peptides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011506:Proteins; D006861:Hydrogen Peroxide