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.
Cardiovascular Research Laboratory, Division of Cardiology, Pulmonology & Vascular Medicine, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany.
Redox Biol. 2018 Jun;16:359-380. doi: 10.1016/j.redox.2018.02.012. Epub 2018 Feb 19.
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.
几种疾病与氧化还原信号的扰动和异常的硫化氢代谢有关，并且存在许多用于测量受影响的含硫物种的分析方法。然而，它们在细胞/生物流体中的浓度和形态仍然存在不确定性，部分原因可能是由于样品处理和检测原理的差异。我们使用超高效液相色谱法结合电喷雾串联质谱法，在这里概述了一种用于同时测量 12 种分析物的特定和敏感平台，包括总硫醇、游离硫醇、其二硫化物和硫化物在血液、唾液和尿液等复杂生物基质中的形态。总测定运行时间<10 分钟，可实现高通量分析。通过利用巯基基团与 N-乙基马来酰亚胺的快速反应，实现了增强的灵敏度和避免人为硫醇氧化。我们优化了分析程序，以检测和分离条件、线性和精密度，包括三个稳定同位素标记的标准。通过实施其他分析物，如甲硫醇、N-乙酰半胱氨酸和辅酶 A，展示了其对未来更全面的硫醇氧化还原代谢组学的多功能性。发现血浆硫化物浓度因样品预处理和烷基化剂的性质而有很大差异。除了以混合二硫化物（S-硫代）的形式与蛋白质结合外，一部分氨基硫醇和硫化物似乎也与蛋白质非共价结合。通过将 10 名健康人类志愿者的血浆氧化还原状态与优化的还原/氧化谷胱甘肽的既定方案进行比较，测试了方法的准确性。在一项原理验证研究中，对包括游离还原/氧化硫醇以及结合的硫醇和硫化物在内的硫醇氧化还原代谢组进行了更深入的分析。在人类血细胞、尿液和唾液中证明了酸不稳定的硫化物/硫醇的额外测定。使用这种简化的基于质谱的工作流程，可以在来自临床和转化研究的样品中确定硫醇氧化还原代谢组，为患者分层、药物监测以及氧化还原疾病的新治疗方法的鉴定提供新的预后/诊断平台。
