Proteome-wide identification of ISG15 sites in HeLa cells

We employed a GlyGly-peptidomics approach to comprehensively map ISG15 modification sites in HeLa cells under three conditions: mock-transfected controls, IFN-induced cells, and cells transfected with the ISGylation machinery. Lysates from wild-type and Isg15 knockout (KO) cells were processed using S-trap, followed by trypsin digestion to generate diglycine-modified peptides. These peptides were enriched via immunoprecipitation and analyzed by LC-MS/MS for identification and quantification. Comparative analysis across conditions and genotypes revealed 671 ISG15 modification sites on 456 distinct proteins, expanding the HeLa ISGylome.