A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome
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/bioflui...
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| Published in: | Redox biology Vol. 16; pp. 359 - 380 |
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| Main Authors: | , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Netherlands
Elsevier B.V
01-06-2018
Elsevier |
| Subjects: | |
| Online Access: | Get full text |
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| Summary: | 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.
•The thiol redox metabolome comprises free and bound thiols, disulfides, and sulfide.•We developed a LC-MS/MS platform for detection of the thiol redox metabolome in biological samples.•Thiols in blood, urine and saliva are stabilized by derivatization with N-ethylmaleimide, also preventing oxidation.•Total and acid-labile thiols are determined following addition of DTT and acid.•Accuracy was investigated by comparison with an established method to assess GSH and GSSG.•Applicability was shown for plasma of 10 healthy volunteers, blood cells, urine and saliva. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally. |
| ISSN: | 2213-2317 2213-2317 |
| DOI: | 10.1016/j.redox.2018.02.012 |