Proteome-wide identification of ISG15 sites targeted by SARS-CoV-2 PLpro

Here, we applied an ISG15-GlyGly peptidomics approach on HeLa cells producing high levels of ISGylated proteins upon stimulation with IFN-α. After obtaining cellular lysates from wild-type and Isg15-/-cells, the samples were incubated with recombinant wild-type or mutant PLpro to catalyze deISGylation of host cellular proteins. After incubation with PLpro, we continued with trypsin digestion to generate diglycine-modified peptides that were subsequently enriched by immunoprecipitation and identified and quantified by LC-MS/MS. By comparing sites across all four conditions, we uncovered 118 ISGylation sites on 95 proteins as targets of the SARS-CoV-2 protease PLpro.