ALKYLATION OF OXYTOCIN BY S-(2-CHLOROETHYL)GLUTATHIONE AND CHARACTERIZATION OF ADDUCTS BY TANDEM MASS-SPECTROMETRY AND EDMAN DEGRADATION

S-(2-Chloroethyl)glutathione (CEG), an alkylating agent formed by glut athione conjugation with 1,2-dichloroethane (DCE), is able to alkylate DNA and proteins. As a prelude to identification of specific protein alkylation sites, the peptide oxytocin was alkylated by CEG, and tande m mass spectrometry was used to identify the alkylation sites. It was found that mono-, bis-, and tris-adducts can result from alkylation of reduced oxytocin and that tandem mass spectrometry differentiated (S- [2-(Cys(1))ethyl]glutathione)oxytocin (mono-adduct Cys-1) from (S-[2-( Cys(6))ethyl]glutathione)oxytocin (mono-adduct Cys-6). Manual Edman de gradation was used to eliminate the possibility that alkylation has oc curred at Tyr-2 rather than at Cys-1 in the case of (S-[2-(Cys(1,6))et hyl]glutathione)oxytocin (bis-adduct) and mono-adduct Cys-1. A mono-ad duct homodimer resulting from alkylation at Cys-6 and disulfide bridge formation through Cys-1 was also identified. Oxidized oxytocin formed two minor adducts, representing less than 5% of the oxytocin present- in the reaction mixture. These findings demonstrate that alkylation of oxytocin by the episulfonium ion of CEG did occur, as evidenced by ta ndem mass spectrometry, and that characterization of these adducts wil l aid in the identification of alkylated amino acids in proteins expos ed to CEG.