Methods in sulfide and persulfide research

Abstract

Sulfides and persulfides/polysulfides (R-S_n-R', n > 2; R-S_n-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides-low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H₂S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H₂S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

Keywords: Hydrogen sulfide; Methodology; Persulfides/polysulfides; Reactive sulfur metabolites; Sulfides.

Fig. 1.

Overview of the MBB method. After derivatization of sulfide, SDB is formed and separated by using RP-HPLC with a gradient elution and is then analyzed by fluorescence detection (excitation wavelength 390 nm, emission wavelength 475 nm).

Fig. 2.

Schematic representation of HPE-IAM-based LC-MS/MS analysis. The LC-ESI-MS/MS approach with HPE-IAM trapping (a) and LC-ESI-MS/MS profiles including each fragmentation pattern (upper panels) of the CysSS_nH standard for each HPE-IAM adduct (b). CysSH, CysSS_nH [i.e. CysSSH and cysteine hydrotrisulfide (CysSSSH)], and other related sulfide compounds are derivatized with HPE-IAM, whose MRM parameters appear in Table 1, and are used for our LC-ESI-MS/MS analysis.

Fig. 3.

Quantitation of the amount of endogenous persulfides/polysulfides in mouse tissues. Data are mean values ± S.D. *p < 0.05, **p < 0.01, ***p < 0.001, N.S., not significant. Student’s t-test.

Fig. 4.

CysSS_nH formation (persulfidation) in various proteins. (a) LC-ESI-MS/MS chromatograms obtained from analysis of CysSS_nH formation in recombinant ADH5 (left panel). The amounts of CysSS_nH detected in ADH5 (right panel). (b) LC-ESI-MS/MS chromatograms obtained from analysis of CysSS_nH in recombinant GAPDH (left panel) and their quantitation (right panel).

Fig. 5.

Reactive sulfur proteome using CN-biotin assay. (a) Schematic illustration of the CN-biotin assay. (b) Protein-cysteine persulfidation identified as spots on membranes transferred from 2D gels. Some labeled spots in the image were subjected to persulfide proteomics, which resulted in identification of several persulfidated proteins.

Fig. 6.

The biotin-PEG-MAL capture method. (a) Schematic illustration of the biotin-PEG-MAL capture method for quantitative identification of endogenous persulfidated proteins, which are isolated by reductive treatment of biotin-PEG₃₆-MAL-bound avidin beads that capture persulfidated proteins. (b) Specific detection with Western blotting (WB) follows use of the capture method. These typical immunoblots show endogenous protein persulfidation of Drp1 (b, left panel) and ADH5 (b, right panel) in HEK293T cells. WT: wild type.

Fig. 7.

Method for detection of specific persulfidated proteins. (a) Schematic illustration of the PMSA for identification of persulfidated proteins by using biotin-PEG₃₆-MAL and different electrophilic compounds. These typical immunoblots show endogenous protein persulfidation of GAPDH (b, upper panels) and ADH5 (b, lower panels) in recombinant protein (b, left panels) and cultured cells (b, right panels). Numbers shown in the gels indicate the numbers of biotin-PEG₃₆-MAL labels in the protein, which indicate the numbers of polysulfidated CysSH residues in the protein.

Fig. 8.

Chemical structures and reactions of the SSP series of fluorescent probes. (a) Structures of SSP2, SSP3, SSP4, and PSP-3. (b, c) Illustration of the reactions of SSP4 (b) and PSP-3 (c) with persulfides.

Fig. 9.

Representative persulfide fluorescence imaging with SSP4 and PSP-3 probes. (a) Illustration of the SSP4 and PSP-3 staining protocol. COS7 cells overexpressing CSE are subjected to fluorescence staining. Na₂S₂ (100 μM) is used as a positive control. (b) Fluorescence images (left panel) and fluorescence intensities (right panel) of SSP4 and PSP-3 staining. Data are mean values ± S.D. *p < 0.05, one-way ANOVA with Tukey’s test.

Fig. 10.

Chemical structure of the QS10 probe and the reaction of QS10 with persulfides for live-cell and real-time imaging of persulfides. Ex., excitation; Em., emission.

Fig. 11.

Representative persulfide fluorescence imaging with the QS10 probe. WT and Cars2 KO HEK293T cells are subjected to fluorescence staining. Fluorescence images (left panel) and a box plot of ratio values for each (right panel). Data are mean values ± S.D. *p < 0.05, Student’s t-test.