Making dataset [MSV000080008] public Adding publication [PMID28035797] to dataset [MSV000080008] Protein ADP-ribosylation is a post-translational modification involved in important physiological and pathological processes. Here, we present an optimized method using the Orbitrap Fusion Tribrid mass spectrometer, which drastically improves the overall ADP-ribosylation site localization score and boosts the identification of ADP-ribosylated peptides. We compared the HCD, ETcaD or EThcD fragmentation methods for the identification of ADP-ribose acceptor sites in a complex cellular sample and further combined them in a product dependent manner, exploiting the unique fragmentation properties of the ADP-ribose moiety as a trigger for a targeted fragmentation of modified peptides.