Plasma Induced Oxidative Cleavage of Disulfide Bonds in Polypeptides during Nanoelectrospray Ionization

Cleavage of the disulfide bond within a polypeptide was observed when the nanoelectrospray (nanoESI) plume of a peptide solution interacted with a low-temperature helium plasma in air. Online mass spectrometric analysis revealed that chain separation accompanied by a mass increase of 1 or 16 Da for each chain was common to peptides having an interchain disulfide bond, while for peptides having intrachain disulfide bonds, the reaction products typically showed mass increases of 17 Da. Experimental results suggested that hydroxyl radicals initiated from the plasma were likely to be responsible via dissociative addition to the disulfide bond (RSSR'), giving rise to RSH and R'SO center dot. When the hydroxyl radical addition product ions ([M + nH + OH](n center dot+), n is the charge state) generated from peptides having intrachain peptides were subjected to collision-induced dissociation (CID) in an ion trap, a-, b-, and y-type sequence ions within the cyclic structure defined by the disulfide bond were observed in addition to the exocyclic cleavages typically seen from CID of [M + nH](n+) peptide ions. Rich structural information could thus be obtained. These findings were demonstrated in 14 peptides containing disulfide bonds and further by bovine insulin, which has three disulfide bonds. Collisional activation of the [M + 5H + OH](5 center dot+) insulin ions provided 76% of the possible backbone cleavages as compared to 26% acquired from CID of the [M + 5H](5+) ions.