Probing disulfide bonding in vivo using immobilized PAO‐affinity chromatography

Protein disulfide bond formation in the reducing cell cytosol has been implicated in redox signaling and catalysis but remains a somewhat controversial and unresolved issue. We have employed a phenylarsine oxide (PAO)‐affinity method improved recently by us to determine intrachain disulfide bonding in vivo in brains from young rats. Proteins containing PAO‐binding vicinal thiols that have been oxidized to disulfide bonds were captured by alkylation of reduced protein thiols and, following removal of the alkylating reagent, reduction of disulfide bonds with tris(2‐carboxyethyl)‐phosphine (TCEP) to promote binding to the immobilized PAO only of the proteins that had contained disulfide bonds. We have begun to identify these proteins by a combination of mass spectrometry and western blotting. We report here that up to 3–5% of proteins from the brain contain PAO‐binding vicinal thiols that are oxidized to disulfide bonds in vivo although the percentage of intracellular proteins containing disulfide bonds is substantially less. Among cytosolic proteins, disulfide bonds were found in a small fraction of the catalytic subunit (PP2Ac) of protein phosphatase 2A and in a much greater fraction of triose phosphate isomerase. These results provide an initial assessment of intrachain disulfide bonding in cytosolic proteins in vivo under presumed healthy conditions.