Forty million or 0.2 g of MCC cells with or without IFN-γ treatment were immunoprecipitated and analyzed by LC-MS/MS (Supplemental Methods). Mass spectra were interpreted using Spectrum Mill software package v7.1 pre-release (Broad Institute) (refs. 35 (link), 71 (link), and Supplemental Methods).
Immunopeptidomes of USP7 inhibitor–treated cell lines were eluted similarly to the method described above, followed by labeling with TMT6 reagent (Thermo Fisher Scientific; XL177A-treated replicates labeled with TMT6-126 and -128; XL177B-treated replicates labeled with TMT6-130 and -131; and WT replicates labeled with TMT6-127 and -129), and then pooled for subsequent fractionation using basic reversed-phase fractionation with increasing concentrations of acetonitrile (10%, 15%, and 50%) in 5 mM ammonium formate (pH 10) and analysis on an Orbitrap Exploris 480 with FAIMS Pro (Thermo Fisher Scientific). Data acquisition parameters were as described with normalized collision energy set to 34% and dynamic exclusion set to 2 seconds.
Immunopeptidomes of USP7 inhibitor–treated cell lines were eluted similarly to the method described above, followed by labeling with TMT6 reagent (Thermo Fisher Scientific; XL177A-treated replicates labeled with TMT6-126 and -128; XL177B-treated replicates labeled with TMT6-130 and -131; and WT replicates labeled with TMT6-127 and -129), and then pooled for subsequent fractionation using basic reversed-phase fractionation with increasing concentrations of acetonitrile (10%, 15%, and 50%) in 5 mM ammonium formate (pH 10) and analysis on an Orbitrap Exploris 480 with FAIMS Pro (Thermo Fisher Scientific). Data acquisition parameters were as described with normalized collision energy set to 34% and dynamic exclusion set to 2 seconds.
Free full text: Click here
