Characterization of the disulfides of bio‐thiols by electrospray ionization and triple‐quadrupole tandem mass spectrometry

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 C--S 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 R--S(+) ions and, in some cases, also the complementary fragments corresponding to the protonated amino acids. Fission across the C--S 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 R--S--S(+) ions are also observed. Fragmentation of glutathione disulfides mainly shows the characteristic loss of the terminal gamma-linked glutamic acid and little, if any, fragmentation of the disulfide system.

Analysis of disulphide linkages in bovine κ‐casein oligomers using two‐dimensional electrophoresis

“…However, the disulphide-linked peptides were preserved using sinapinic acid in linear mode. We also observed asymmetric cleavage of the disulphide bond during CID following ESI as reported previously with CID [15,18] and PSD [16]. Generally the disulphide-linked peptides we examined exhibited relatively poor fragmentation requiring high collision energies especially for the large multivalent ions of interest in this study.…”

Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

“…An important feature of all the prominent product ions for GSSG is the preservation of the disulfide bond. Two minority product ions, m/z = 308 and 306, exhibit S-S bond cleavage, which has been observed previously [34]. (Table 2).…”

A novel salt bridge mechanism highlights the need for nonmobile proton conditions to promote disulfide bond cleavage in protonated peptides under low-energy collisional activation

“…However, it is expected that the intramolecular proton transfer process to be lower in energy than bond cleavage processes. It should be noted that Structure F is related to one of the neutral structures previously proposed°for°S-S°bond°cleavage°products° [19].…”

“…Thus, the absence of a mobile proton [17,18] appears to facilitate disulfide bond cleavage. The mechanistic features of these bond cleavage reactions have not been well established, although we were intrigued by a recent report [19], which proposed that S-S bond cleavage leads to the formation of a sulfenylium ion (R-S ϩ ) and that C-S bond cleavage forms the R-S-S ϩ product ion. Given that RS ϩ cations are not very stable [20], the formation of cyclic product ions via neighboring group processes [6] seemed more likely.…”