Two complementary experiments were performed to evaluate whether N8-propargyl spermidine is a substrate of deoxyhypusine synthase in cells. HEK293 and mouse embryonic fibroblast (MEF) cells were incubated with either N8-propargyl spermidine (Probe 1) or spermidine, lysed, and subjected to CuAAC ligation with azido-PEG3-biotin. In the MEF experiment, biotinylated proteins were enriched on NeutrAvidin agarose resin, digested on-bead with trypsin, and, following TMT labeling and LC-MS/MS analysis, the data were processed in MaxQuant and analyzed for intensity enrichment in samples derived from cells treated with Probe 1 versus spermidine. In the HEK293 experiment, proteins were first digested with trypsin, after which biotinylated peptides were enriched on NeutrAvidin agarose resin and analyzed by LC-MS/MS. During MaxQuant processing, the peptide-level data were searched for Probe 1-derived hypusine and deoxyhypusine modifications carrying the biotinylated triazole tag, as well as for Probe 1-derived thiotriazole adducts on cysteine residues.