A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome

Abstract

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Speciation of reactive sulfur species and their reactions with alkylating agents: do we have any clue about what is present inside the cell?

“…This clearly indicates that MBB can shift the speciation of sulfur species in human serum and plasma and extract sulfide from different biological sulfide pools as we proposed previously (see Nagy et al ., ). A previous study found that more sulfide is detected in whole blood when using NEM or MBB compared with when using IAM, which is consistent with our proposal that NEM and MBB can more effectively cleave polysulfur chains than IAM (Sutton et al ., ).…”

“…Similar reactions may occur with the corresponding NEM polysulfide adducts. Such a behaviour would also be consistent with another recent observation, that is, that the NEM adduct of S 2 2− is not stable at pH 7.4 (Sutton et al ., ), supporting the notion that NEM‐derivatized and IAM‐derivatized polysulfide adducts are metastable and both, NEM‐S and IAM‐S are good leaving groups. We used DMPO trying to capture sulfur‐centred products generated upon homolytic cleavage of GSH polysulfide species under two different conditions (using alkylated polysulfides and non‐alkylated polysulfides) but were unable to detect DMPO adducts under any experimental condition (see ).…”

Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues

“…While it has been proposed that addition of NEM in higher concentrations to blood tubes before plasma preparation can potentially lead to leakage of GSH-NEM from the RBCs [10], our final NEM concentration (2.5 mM) was well below the range where leakage was observed. Delays between sample collection and NEM addition resulted in a reduction of measured reduced-to-oxidized-glutathione ratios in serum and plasma but not in whole blood (Figure 4).…”

“…While it has been proposed that addition of NEM in higher concentrations to blood tubes before plasma preparation can potentially lead to leakage of GSH-NEM from the RBCs [10], our final NEM concentration (2.5 mM) was well below the range where leakage was observed.…”

“…However, as the cellular GSH concentration is approximately two orders of magnitude higher than the GSSG concentration, postsampling oxidation of even a small fraction of GSH to GSSG can lead to a severe bias of the concentration measured for the latter. In order to prevent artificially high GSSG levels, it is now widely accepted that all biological samples should be treated with a thiol-quenching reagent which captures free GSH immediately after sampling and prior to further sample processing [9,10]. The most commonly used GSH conjugating reagent is N-ethylmaleimide (NEM) due to its cell permeability and fast reaction kinetics.…”