Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography-mass spectrometry.

S-methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O-6-methyl-guanine to revert the normal GC base pairing. To assess the value of SMC and of S-methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC-MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra- and inter-individual variability. Extractive alkylation with butylchloroformate-n-butanol-pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC-MS with S-[CD(3) ]-SMC and -SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium-polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile-age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02-0.7 µg/mL range (n = 61) with large inter-day and inter-individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90-810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes.

Toward an "omic" physiopathology of reactive chemicals: thirty years of mass spectrometric study of the protein adducts with endogenous and xenobiotic compounds.

Cancer and degenerative diseases are major causes of morbidity and death, derived from the permanent modification of key biopolymers such as DNA and regulatory proteins by usually smaller, reactive molecules, present in the environment or generated from endogenous and xenobiotic components by the body's own biochemical mechanisms (molecular adducts). In particular, protein adducts with organic electrophiles have been studied for more than 30 [see, e.g., Calleman et al., 1978] years essentially for three purposes: (a) as passive monitors of the mean level of individual exposure to specific chemicals, either endogenously present in the human body or to which the subject is exposed through food or environmental contamination; (b) as quantitative indicators of the mean extent of the individual metabolic processing which converts a non-reactive chemical substance into its toxic products able to damage DNA (en route to cancer induction through genotoxic mechanisms) or key proteins (as in the case of several drugs, pesticides or otherwise biologically active substances); (c) to relate the extent of protein modification to that of biological function impairment (such as enzyme inhibition) finally causing the specific health damage. This review describes the role that contemporary mass spectrometry-based approaches employed in the qualitative and quantitative study of protein-electrophile adducts play in the discovery of the (bio)chemical mechanisms of toxic substances and highlights the future directions of research in this field. A particular emphasis is given to the measurement of often high levels of the protein adducts of several industrial and environmental pollutants in unexposed human populations, a phenomenon which highlights the possibility that a number of small organic molecules are generated in the human organism through minor metabolic processes, the imbalance of which may be the cause of "spontaneous" cases of cancer and of other degenerative diseases of still uncharacterized etiology. With all this in mind, it is foreseen that a holistic description of cellular functions will take advantage of new analytical methods based on time-integrated metabolomic measurements of a new biological compartment, the "adductome," aimed at better understanding integrated organism response to environmental and endogenous stressors.

Thiol-disulfide redox equilibria of glutathione metaboloma compounds investigated by tandem mass spectrometry.

The thiol group of cysteine plays a pivotal role in structural and functional biology. We use mass spectrometry to study glutathione-related homo- and heterodimeric disulfides, aiming at understanding the factors affecting the redox potentials of different disulfide/thiol pairs. Several electrospray ionization (ESI)-protonated disulfides of cysteamine, cysteine, penicillamine, N-acetylcysteine, N-acetylpenicillamine, gammaGluCySH, HSCyGly, and glutathione were analyzed on a triple quadrupole instrument to measure their energy-resolved tandem mass spectra. Fission of the disulfide bond yields RSH*H(+) and RS(+) ions. The logarithm of the intensity ratio of the RS(+)/RSH*H(+) fragments in homodimeric disulfides is proportional to the normal reduction potential of their RSSR/RSH pairs determined by nuclear magnetic resonance (NMR) in solution, the more reducing ones yielding the higher ratios. Also in some R(1)S-SR(2) disulfides, the ratio of the intensities of the RSH + H(+) and RS(+) ions of each participating thiol shows a linear relationship with the Nernst equation potential difference of the corresponding redox pairs. This behavior allows us to measure the redox potentials of some disulfide/thiol pairs by using different thiol-reducing probes of known oxidoreductive potential as reference. To assist understanding of the fission mechanism of the disulfide bond, the fragments tentatively identified as 'sulfenium' were themselves fragmented; accurate mass measurement of the resulting second-generation fragments demonstrated a loss of thioformaldehyde, thus supporting the assigned structure of this elusive intermediate of the oxidative stress pathway. Understanding this fragmentation process allows us to employ this technique with larger molecules to measure by mass spectrometry the micro-redox properties of different disulfide bonds in peptides with catalytic and signaling biological activity.

A study of the glutathione metaboloma peptides by energy-resolved mass spectrometry as a tool to investigate into the interference of toxic heavy metals with their metabolic processes.

To better understand the fragmentation processes of the metal-biothiol conjugates and their possible significance in biological terms, an energy-resolved mass spectrometric study of the glutathione conjugates of heavy metals, of several thiols and disulfides of the glutathione metaboloma has been carried out. The main fragmentation process of gamma-glutamyl compounds, whether in the thiol, disulfide, thioether or metal-bis-thiolate form, is the loss of the gamma-glutamyl residue, a process which ERMS data showed to be hardly influenced by the sulfur substitution. However, loss of the gamma-glutamyl residue from the mono-S-glutathionyl-mercury (II) cation is a much more energetic process, possibly pointing at a strong coordination of the carboxylic group to the metal. Moreover, loss of neutral mercury from ions containing the gamma-glutamyl residue to yield a sulfenium cation was a much more energetic process than those not containing them, suggesting that the redox potential of the thiol/disulfide system plays a role in the formal reduction of the mercury dication in the gas phase. Occurrence of complementary sulfenium and protonated thiol fragments in the spectra of protonated disulfides of the glutathione metaboloma mirrors the thiol/disulfide redox process of biological importance. The intensity ratio of the fragments is proportional to the reduction potential in solution of the corresponding redox pairs. This finding has allowed the calculation of the previously unreported reduction potentials for the disulfide/thiol pair of cysteinylglycine, thereby confirming the decomposition scheme of bis- and mono-S-glutathionyl-mercury (II) ions. Finally, on the sole basis of the mass spectrometric fragmentation of the glutathione-mercury conjugates, and supported by independent literature evidence, an unprecedented mechanism for mercury ion-induced cellular oxidative stress could be proposed, based on the depletion of the glutathione pool by a catalytic mechanism acting on the metal (II)-thiol conjugates and involving as a necessary step the enzymatic removal of the glutamic acid residue to yield a mercury (II)-cysteinyl-glycine conjugate capable of regenerating neutral mercury through the oxidation of glutathione thiols to the corresponding disulfides.

Electrospray ionization and triple quadrupole tandem mass spectrometry study of some biologically relevant homo- and heterodimeric cadmium thiolate conjugates.

A series of 19 compounds of general formula R1S-Cd-SR2, R1, and R2, being some biologically relevant thiol amino acids and peptides, were prepared by direct reaction of cadmium(II) ions and thiols in water at millimolar concentration. The obtained products were characterized by electrospray ionization and triple quadrupole tandem mass spectrometry. The source spectra of stoichiometric 1:2 Cd-thiol systems containing either an individual thiol or equimolar mixtures of two different thiols featured several Cd-containing signals, although at much lesser intensity than in the previously reported experiments with mercury(II) (J. Am. Soc. Mass Spectrom. 2004, 15, 288-300). Also, the relative intensity of the homo- and heterodimeric thiolates were significantly different from the theoretically expected 1:2:1 ratio, thus pointing at some degree of discrimination between the different thiols. In particular, homo-cysteine showed much less reactivity than cysteine, and penicillamine and cysteine methyl ester much less than the free amino acid. The fragment spectra show structure-specific ions for the different ligands bound to the metal ion and allow a stand-alone determination of the connectivity also of isomeric pairs. The fragmentation pathways are similar to those observed for the corresponding mercury(II) analogues, with the addition of further intense and specific fragments, one formally carrying a Cd-bound OH ligand and one connected as a five-membered oxazolone carrying a cadmium-bis-thiolate side chain, both formed with a high intensity. Energy-resolved fragmentation data show that metal-free ions can be generated from cysteine but not from glutathione conjugates and point to the possibility of unveiling differences in the biochemical behavior of the conjugates of different heavy metals through the detailed study of their mass spectrometric fragmentation.