Synthesis of the desthiobiotin-phenylarsenic oxide probe for identification and analysis of arsenic-binding proteins in MCF-7 cells.

A novel organoarsenic probe, desthiobiotin-phenylarsenic oxide (DesBiotin-PAO(III)), was synthesized by coupling desthiobiotin with p-aminophenylarsenic acid (PAO(III)) via a mixed anhydride method. This probe enables the in situ capture of intracellular arsenic binding proteins, which are subsequently adsorbed onto streptavidin-coated magnetic beads (SA-MBs) through the desthiobiotin-streptavidin interaction, thereby achieving selective separation of arsenic-binding proteins from other intracellular components. Because the binding affinity of desthiobiotin for streptavidin is considerably weaker than that of biotin, free biotin can be employed as a competitive displacement agent to gently release the captured arsenic-binding proteins from the SA-MBs. Comprehensive characterization of the synthesized probe confirmed the successful preparation of DesBiotin-PAO(III). Using reduced bovine serum albumin (rBSA) as a model arsenic-binding protein, the capture performance of DesBiotin-PAO(III) was systematically validated. Building on this, DesBiotin-PAO(III) was employed for the in situ capture of arsenic-binding proteins in MCF-7 cells, and the captured proteins were identified by high performance liquid chromatography (HPLC)- electrospray ionization tandem mass spectrometry (ESI-MS/MS) (separated using C18 column with gradient elution (H2O-CH3CN containing 0.1% formic acid) and detected in positive mode). In total, 153 arsenic-binding proteins were ultimately identified in MCF-7 cells. Compared with previously reported methods based on organoarsenic probes, the present approach identified the greatest number of arsenic-binding proteins to date.