| Biotin switch assay |
This was the first assay developed for the detection of persulfidation. It was based on the premise that persulfides would not react with electrophilic thiolblocking reagent S-methyl methanethiosulfonate (MMTS). Persulfides were subsequently labeled with N-[6-(biotinamido)hexyl]- 30 -(20 -pyridyldithio)propionamide (biotin-HPDP). The biotin labeled species were pulled down by streptavidin beads and analysed by MS. It was demonstrated that upto 25% of proteins in the liver were persulfidated under basal conditions. The chemistry was later on proved to be incorrect as persulfides were shown to react with both electrophilic and nucleophilic species. |
(103) |
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| IAA assay |
According to this assay, Iodoacetic acid (IAA) a thiol blocking agent would react with both free thiols and protein persulfides. Subsequently, DTT would cleave the alkylated persulfide. This was followed by labeling of that particular cysteine with iodoacetamide-linked biotin (IAP). However, how this method distinguishes the persulfides from intramolecular and intermolecular disulfides and S-nitrosothiols, which would also be reduced by DTT, remains poorly understood. |
(130) |
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| Maleimide assay |
This assay was based on the fact that N-ethyl maleimide, a thiol-blocking agent, would block both free thiol and persulfide. Maleimide was conjugated to Cy5. DTT was used to cleave the alkylated moieties. Fluorescence signal would decrease if the sample contains persulfides. Ratiometric decrease in fluorescence can be used for quantification. |
(131) |
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| Biotin thiol assay |
Maleimide-PEG2-Biotin was used to alkylate both thiols and persulfides followed by binding of the proteins on an avidin column. Elution was done by DTT which cleaved the disulfide bond leaving the biotin tag bound to the column. The eluate containing the persulfidated proteins was analysed by Western blotting and LC-MS/MS. |
(132) |
|
| Pro-Per-DP –Protein persulfide detection protocol |
Iodoacteyl PEG2 Biotin was used to alkylate both free thiols and persulfides. These species were pulled down by streptavidin coated magnetic beads and subsequently reduced with DTT. The supernatant, containing the persulfidated proteins were analysed by MS. |
(133) |
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| Tag switch assay |
This assay exploits the difference in chemical and physical properties of the thiol and the persulfide group. A thiol-blocking reagent methylsulfonyl benzothiazole, reacts with free thiol and persulfide. When compared to disulfides in proteins, the disulfide bond in persulfide adducts react strongly to nucleophiles. Subsequently, a new tag-switch reagent, biotinylated cyanoacetic acid was used to label persulfidated proteins specifically. This method has been further improved by the use of cyanoacetic acid derivatives with BODIPY moiety (CN-BOT) for labeling cells and Cy3-dye (CN-Cy3) for labeling cell lysates, respectively. |
(102,134) |
