Updated project metadata. We developed a strategy that allows the identification of NEDP1 dependent NEDDylation sites under endogenous expression of wild type NEDD8. We combined the use of anti-diGly antibodies that recognise both ubiquitin and NEDD8 modified peptides upon trypsin digestion with short treatment of cells with the ubiquitin E1 inhibitor MLN7243 (UAEi), that dramatically reduces ubiquitin but not NEDD8 modification. By eliminating the majority of ubiquitin-derived diGly peptides upon MLN7243 treatment, we would be able to quantify NEDP1 dependent diGly peptides. Extracts from parental and NEDP1 knockout HCT116 cells both treated with UAEi were used for the isolation of diGly modified peptides and mass spectrometry analysis.