HeLa cell protein interactome of polyamine affinity-based probes.

HeLa cells were incubated with affinity-based probes designed as synthetic analogs of endogenous metabolites: putrescine, spermidine, and spermine, while a monoamine compound served as a control. Probe-protein interactions were captured in situ using UV-induced crosslinking. After cell lysis, biorthogonal ligation and affinity purification of the probe-conjugated proteins were carried out. These proteins were digested with trypsin, and the resulting peptides were labeled with TMT. Proteins that showed enrichment in samples treated with polyamine probes, compared to those treated with the monoamine control, were identified as polyamine-binding proteins.