New Quantitative Mass Spectrometry Approaches Reveal Different ADP-ribosylation Phases Dependent On the Levels of Oxidative Stress [Technological Innovation and Resources]

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.

from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2qnRc9L
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