4.9. Chemical Proteomics

A chemical proteomic approach, able to define cellular targets of selected drug candidates based on compound-immobilized affinity chromatography, was used as previously described [58,59] to identify potential molecular interactions of IPA. To that aim, cytokinin was biotynilated through a two-step modification procedure; briefly, IPA was incubated with N(3Br-propyl)phthalimide in N,N-dimethylformamide in the presence of 1 mM NaH. The resulting product was than underwent hydrazinolysis, followed by incubation with biotin-aldehyde. The product was purified by HPLC-UV and its structure and purity were confirmed by mass spectrometry. DLD1 were incubated with 10 μM biotinylated IPA for two hours at 37 °C and then underwent protein extraction procedure. Subcellular fractionation of nuclear and cytosolic protein from treated and control cells was obtained using NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) as described previously [53]. Clarified whole cell lysates (100 μg) were incubated with IPA-loaded or with control resin (for 2 h at 25 °C) and interacting proteins were eluted and analyzed as reported elsewhere [58].