Negative Ion Tandem Mass Spectrometry for the Detection of Glutathione Conjugates

Abstract

Characterization of S-linked conjugates of the endogenous tripeptide glutathione (gamma-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC-MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the [M - H]- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated gamma-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.

Nucleophile addition of reduced glutathione on 2-methyl-2-nitroso compound: A combined electron paramagnetic resonance spectroscopy and electrospray tandem mass spectrometry study

“…However, dissociation reactions observed for [1b ϩ H] ϩ remain poorly informative. In contrast, data obtained while submitting the deprotonated 1b molecule, [1b Ϫ H] -, to CID (Figure 1b) allows the structure proposed for this hydroxylamine to be validated, based on fragmentation rules recently established for glutathione conjugates ionized in the negative mode [19]. Since the dissociation pathways of such deprotonated molecules have already been thoroughly discussed by Dieckhaus et al [19], they will only be briefly described here.…”

“…In contrast, data obtained while submitting the deprotonated 1b molecule, [1b Ϫ H] -, to CID (Figure 1b) allows the structure proposed for this hydroxylamine to be validated, based on fragmentation rules recently established for glutathione conjugates ionized in the negative mode [19]. Since the dissociation pathways of such deprotonated molecules have already been thoroughly discussed by Dieckhaus et al [19], they will only be briefly described here. The intense m/z 272.1 product ion in Figure 1b [19], it is shown here that neither the cation nor the anion obtained after electrospray of the same species follows some common trends.…”

“…As a result, detection of glutathione conjugates is often performed using a 129 Da neutral loss MS/MS experiments [13][14][15][16][17]. Nevertheless, different classes of glutathione conjugates, such as aliphatic thioether adducts ionized in the positive ion mode, were shown to behave differently upon collisioninduced dissociation (CID) [18,19]. An alternative approach was reported using the negative ion mode with a characteristic MS/MS reaction, yielding the deprotonated ␥-glutamyl-dehydroalanyl-glycine at m/z 272, shown to occur for any glutathione conjugates [19].…”

“…Nevertheless, different classes of glutathione conjugates, such as aliphatic thioether adducts ionized in the positive ion mode, were shown to behave differently upon collisioninduced dissociation (CID) [18,19]. An alternative approach was reported using the negative ion mode with a characteristic MS/MS reaction, yielding the deprotonated ␥-glutamyl-dehydroalanyl-glycine at m/z 272, shown to occur for any glutathione conjugates [19]. The usefulness of this approach was demonstrated in a variety of GSH adducts obtained from the reaction between reactive metabolites and this tripeptide [19].…”

“…An alternative approach was reported using the negative ion mode with a characteristic MS/MS reaction, yielding the deprotonated ␥-glutamyl-dehydroalanyl-glycine at m/z 272, shown to occur for any glutathione conjugates [19]. The usefulness of this approach was demonstrated in a variety of GSH adducts obtained from the reaction between reactive metabolites and this tripeptide [19].…”