Abstract
Arsenic is a widespread environmental contaminant, and long-term exposure to arsenic in drinking water is known to be associated with the development of many human diseases. Identification of arsenic-binding proteins is important for understanding the mechanisms underlying the toxic effects of arsenic species. Here, we developed a chemoproteomic strategy, relying on the use of a biotin-As(III) probe, stable isotope labeling by amino acids in cell culture, and liquid chromatography-tandem mass spectrometry analysis, to identify quantitatively As(III)-binding proteins. Over 400 proteins were enriched from the lysate of HEK293T cells with streptavidin beads immobilized with the biotin-As(III) probe. Competitive labeling experiments in the presence or absence of p-aminophenylarsenoxide (PAPAO) revealed 51 candidate As(III)-binding proteins, including several molecular chaperones and cochaperones, that is, HSPA4, HSPA4L, HSPH1, HOP1, FKBP51, and FKBP52. We also validated, by employing western blot analysis, the ability of HSPA4, a member of heat shock protein 70 (HSP70) family, in binding with PAPAO and sodium arsenite in vitro. Together, our work led to the identification of a number of new As(III)-interaction proteins, and our results suggest that As(III) may perturb proteostasis partly through binding directly with molecular chaperones.