Evaluation of N8-propargyl spermidine as a substrate of deoxyhypusine synthase in cellulo

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 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 N8-propargyl spermidine versus spermidine. In the HEK293 experiment, proteins were digested with trypsin, biotinylated peptides were enriched on NeutrAvidin agarose resin, and after LC-MS/MS analysis and MaxQuant processing, the peptides were examined for the presence of N8-propargyl spermidine thio-triazole adducts on cysteine residues.