New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS - PubMed (original) (raw)
Bo Tan 1 2 3, Jiping Sun 5, Zhongkai Gu 6 7, Xiaotian Sun 8, Yichun Zhu 5, Keke Huo 7, Zonglian Cao 9, Ping Yang 9, Xiaoming Xin 3, Xinhua Liu 3, Lilong Pan 3, Furong Qiu 1, Jian Jiang 1, Yiqun Jia 10, Fuyuan Ye 10, Ying Xie 1 2, Yi Zhun Zhu 2 3
Affiliations
- PMID: 28406238
- PMCID: PMC5390247
- DOI: 10.1038/srep46278
New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS
Bo Tan et al. Sci Rep. 2017.
Abstract
Hydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method for the determination of hydrogen sulfide in various biological matrices by determination of a derivative of hydrogen sulfide and monobromobimane named sulfide dibimane (SDB). 36S-labeled SDB was synthesized and validated for using as an internal standard. This method has been successfully used to measure hydrogen sulfide levels in a broad range of biological matrices, such as blood, plasma, tissues, cells, and enzymes, across different species. Moreover, a novel mode that hydrogen sulfide could loosely and non-covalently bind to human serum protein (HSA) and hemoglobin (HB) was revealed by using the developed method.
Conflict of interest statement
The authors declare no competing financial interests.
Figures
Figure 1. Analysis of hydrogen sulfide in different biological matrices.
(a,b) The MS/MS spectrum of hydrogen sulfide derivatives, sulfide dibimane (SDB) (a) and 36S-labeled sulfide dibimane (36S-SDB) (b). (c,d) The selected reaction monitoring (SRM) chromatograms (m/z 415.3 to m/z 223.3) (c) or (m/z 419.3 to m/z 227.3) (d) for the sodium sulfide solution (5 μM). (e,f) The SRM chromatograms (m/z 415.3 to m/z 223.3) (e) or (m/z 419.3 to m/z 227.3) (f) for the 36S labeled-sodium sulfide solution (5 μM). (g) The combined SRM chromatograms of SDB (1.25 μM) and 36S-labeled SDB (0.313 μM) in an authentic standard solution. (h) The combined SRM chromatograms of SDB and 36S-labeled SDB (IS) in mice blank plasma. Data were collected using UPLC (Waters) coupled with a triple quadrupole mass spectrometer (AB Sciex 5500).
Figure 2. Optimization of MBB derivatization conditions.
(a,b) Test for three pH values and incubation times in a sodium sulfide solution (1 μM) (a) and mice plasma (b). (c) Normalized responses at three pH values by the responses at pH 7.5 for either a sodium sulfide solution (1 μM) or mice plasma after 120 min of derivatization. (d) Comparison of the response for Tris-HCl (pH 8.5) buffer at three ionic strengths (100, 200, 600 mM) in mice plasma. (e) Comparison of linearity of response of four sodium sulfide levels (0.313, 1.25, 5 and 20 μM) with or without 2.0 mg/mL EDTA. Data are represented as the mean ± SD (n = 3).
Figure 3. Validation of the robustness, signal-to-noise ratio and linearity of the assay.
(a) Variance of responses for hydrogen sulfide in processed mice plasma without the internal standard (IS) over 24 h. (b) Variance of responses for hydrogen sulfide in processed mice plasma with the IS over 30 days. (c) The typical standard curve for hydrogen sulfide with the IS (y = 2.57_x_ + 0.025, r = 0.9981). (d) Comparison of external standard curve between hydrogen sulfide and 36S-labeled hydrogen sulfide. (e) The signal-to-noise value for hydrogen sulfide at the lower limit of quantification (0.039 μM). (f) Stability of hydrogen sulfide in processed mice plasma under room light for 24 h (data were reconstructed by the IS response). Each point represents the average value of the triplicates. The dotted line represents ± 15% variation.
Figure 4. Determination of hydrogen sulfide in rat cardiac ventricular myocytes (NRCMs) and yeast CSE enzyme (CYS3).
(a) Hydrogen sulfide production with two substrates, L-cysteine for CSE and D-cysteine for 3MST in NRCMs. (b) Hydrogen sulfide production with or without CSE siRNA in NRCMs. (c) Hydrogen sulfide levels with or without high glucose treatment in NRCMs. (d) Hydrogen sulfide production with L-cystathionine (CTT) in yeast CYS3. (e,f) Hydrogen sulfide production with S-propargyl-cysteine (SPRC) in yeast CYS3 without (e) or with (f) the propargylglycine (PAG) inhibitor.
Figure 5. Determination of hydrogen sulfide in mice tissues.
(a) Hydrogen sulfide concentration in different tissues in both wild-type mice (CSE +/+) and CSE knockout mice (_CSE_−/−). (b) Hydrogen sulfide concentration in different tissues in wild-type mice with propargylglycine (PAG, CSE inhibitor) or amino-oxyacetate (AOA, CBS inhibitor) treatment. (c) Hydrogen sulfide concentrations in rat plasma after a single intraperitoneal injection of 12.5, 25 and 50 μM NaHS solution using the LC-MS-MS or HPLC-FL method. * represents P < 0.05; ** represents P < 0.01; ns represents not significant.
Figure 6. Determination of hydrogen sulfide in human blood (n = 6).
(a) The baseline hydrogen sulfide in human blood, plasma, and red cells. (b) The hydrogen sulfide levels in human blood, plasma, and red cells pretreated with DTT. (c) The DTT reducible hydrogen sulfide levels in human blood, plasma, and red cells. (d) The ratio between calculated hydrogen sulfide and determined hydrogen sulfide levels in human blood with or without DTT. (e) The protein binding rate of hydrogen sulfide in human plasma pretreated with or without DTT. (f) The typical blank subtracted sensorgrams of hydrogen sulfide to human serum protein (HSA) with the hydrogen sulfide levels ranging from 0 to 10 mM. (g) The typical blank subtracted sensorgrams of hydrogen sulfide to human hemoglobin (HB) with the hydrogen sulfide levels ranging from 0 to 10 μM. Data are represented as the mean ± SD, except for the sensorgrams. (h) The blank subtracted sensorgrams of (S)-warfarin to human serum protein (HSA) with hydrogen sulfide levels ranging from 0 to 64 nM.
References
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