Chemoproteomic Approach for the Quantitative Identification of Arsenic-Binding Proteins

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.