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.
Highlights
Many biological processes that have previously been associated with an overproduction of reactive oxygen species (ROS) and/or an impaired antioxidant and free radical scavenging capacity were thought to culminate in ‘oxidative stress’, cell death and tissue damage
There is no shortage of analytical methods for the determination of biological thiols, and many more assays appeared in just the last couple of years
Several of those more recent additions to our analytical armamentarium use a combination of some form of chromatographic separation (HPLC, ultrahigh performance liquid chromatography (UPLC) or capillary electrophoresis) and mass-spectrometry for detection
Summary
Many biological processes that have previously been associated with an overproduction of reactive oxygen species (ROS) and/or an impaired antioxidant and free radical scavenging capacity were thought to culminate in ‘oxidative stress’, cell death and tissue damage Such conditions are interpreted to reflect situations in which a shift in redox poise has occurred, affecting both global and regional. There is an increased interest in analytical methods that provide a more refined mapping of the associated metabolic changes and enable further study of the underlying mechanisms In parallel with those developments, there has been a resurgence of interest in the biological effects of hydrogen sulfide (H2S) and related oxidation products such as persulfides and polysulfides [4,5,6]. Sulfide is involved in post-translational protein modification (persulfidation, S-sulfhydration) whereby reactive cysteine groups involved in redox signaling are modified, resulting in altered chemical reactivity and protein function [7]
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