A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome
T.R. Sutton,
M. Minnion,
F. Barbarino,
G. Koster,
B.O. Fernandez,
A.F. Cumpstey,
P. Wischmann,
M. Madhani,
M.P. Frenneaux,
A.D. Postle,
M.M. Cortese-Krott,
M. Feelisch
Affiliations
T.R. Sutton
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
M. Minnion
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
F. Barbarino
Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology & Vascular Medicine, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany
G. Koster
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
B.O. Fernandez
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
A.F. Cumpstey
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
P. Wischmann
Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology & Vascular Medicine, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany
M. Madhani
Institute of Cardiovascular Sciences, University of Birmingham, Birmingham, UK
M.P. Frenneaux
Norwich Medical School, University of East Anglia, Norwich, UK
A.D. Postle
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK
M.M. Cortese-Krott
Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology & Vascular Medicine, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany
M. Feelisch
Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton; NIHR Southampton Biomedical Research Centre, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK; Correspondence to: Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital, South Academic Block, Level F, Mailpoint 810, Tremona Road, Southampton SO16 6YD, UK.
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. Keywords: Oxidative stress, Redox status, Reactive species interactome, Glutathione, Hydrogen sulfide, Persulfides, Thiol-maleimide michael addition
