We employed an AP-MS approach to map ISG15 modification sites on GAPDH and PGK1 in HEK293T cells by overexpression of FLAG-GAPDH, FLAG-PGK1 or FLAG-GFP together with the ISG15 conjugation enzymes (E1, E2, E3) and wild-type HA-ISG15 or conjugation-defective ISG15-AA. Cells were lysed in physiological buffer, subjected to FLAG-pulldown followed by on-bead digestion and further processing for LC-MS/MS analysis.