Protein ISGylation profiles in human cells have been previously reported, but with limited depth and predominantly based on overexpression systems.30–35 To obtain deep coverage and modified residue information in a more physiologically relevant context, we decided to combine advanced proteomic techniques with the immunoaffinity purification of GlyGly tryptic peptides36 in wild-type (WT) and USP18−/− knockout (KO) HAP1 cells, in order to analyse the first USP18-dependent ISGylome of human cancer cells. This evidence concerns the gene USP18 and cancer.