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

Research output: Contribution to journalArticle

Authors

  • T. R. Sutton
  • M. Minnion
  • F. Barbarino
  • G. Koster
  • B. O. fernandez
  • A. F. Cumpstey
  • P. Wischmann
  • M. P. Frenneaux
  • A. D. Postle
  • M. M. Cortese-Krott
  • M. Feelisch

Colleges, School and Institutes

External organisations

  • Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton
  • University Hospital Southampton NHS Foundation Trust
  • Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology & Vascular Medicine, Medical Faculty, Heinrich Heine University
  • University of East Anglia

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.

Details

Original languageEnglish
Pages (from-to)359-380
JournalRedox Biology
Volume16
Early online date19 Feb 2018
Publication statusPublished - 1 Jun 2018

Keywords

  • oxidative stress , redox status , reactive species interactome , glutathione , hydrogen sulfide , persulfides , thiol-maleimide michael addition