Aston Labs

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.   On-line mass spectrometric analysis revealed that chain separation accompanied by a mass increase of 1 Da or 16 Da for each chain was common to peptides having an inter-chain disulfide bond, while for peptides having intra-chain 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^•.  When the hydroxyl radical addition product ions ([M+nH+OH]^n•+, n: charge state) generated from peptides having intra-chain 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 together with improved ability to locate the disulfide bond due to the mass tags automatically added onto the cleavage sites.  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•+ insulin ions, formed in situ by reaction with hydroxyl radicals, provided 76% of the possible backbone cleavages as compared to 26% acquired from CID of the [M+5H]⁵⁺ ions.