Abstract
Glutathione and other intracellular low molecular mass thiols act both as the major endogenous antioxidant and redox buffer system and, as recently highlighted, as an important regulator of cellular homeostasis. Such cellular functions are mediated by protein thiolation, a newly recognized post‐translational modification which involves the formation of mixed disulfides between GSH and key disulfide‐linked Cys residues in the native protein structure. It is also well known that thiol‐seeking heavy metals, such as mercury, cadmium and lead, may interfere in this regulatory system, thus disrupting the cellular functioning. To identify such mixed disulfides in order to investigate their biological role, 15 homo‐ and heterodimeric disulfides were prepared by air oxidation of binary mixtures containing cysteine, homocysteine, penicillamine, N‐acetylcysteine, N‐acetylpenicillamine and glutathione and their protonated molecules were characterized by mass spectrometry. Collisionally activated unimolecular decomposition of protonated homo‐ and heterodimeric disulfides generated by electrospray ionization gives rise to fission of the disulfide system both between the two sulfur atoms and across the CS bonds, to yield structurally specific fragments which allow one to define the structure of the compounds and to discriminate between isomeric compounds. Fission between the sulfur atoms yields a pair of RS^+^ ions and, in some cases, also the complementary fragments corresponding to the protonated amino acids. Fission across the CS bonds mainly occurs in the disulfides of N‐acetylcysteine and N‐acetylpenicillamine and gives rise to non‐S‐containing fragments formally similar to those obtained from some mercapturic acids. The complementary fragments, formally connected as RSS^+^ ions are also observed. Fragmentation of glutathione disulfides mainly shows the characteristic loss of the terminal γ‐linked glutamic acid and little, if any, fragmentation of the disulfide system. Copyright © 2004 John Wiley & Sons, Ltd.