Investigating protein thiol chemistry associated with dehydroascorbate, homocysteine and glutathione using mass spectrometry

Abstract

Rationale:Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigate S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. Methods:Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) andGrx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. Results: ESI-MS analysis reveals that DHA mediates disulfide formation andS-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1, HHb and Prdx2.The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. Conclusions:Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

Peroxiredoxins wear many hats: Factors that fashion their peroxide sensing personalities

“…For example, AFM was used to film biological molecules and examine the oligomeric state of Prdx at 10–16 frames/s, while EFM revealed the formation of nanorods containing Fe 2+ in the central cavity as a result of the self-assembly of Prdx. [ 136 , 139 , 141 , 142 ] Mass spectrometry (MS): electrospray ionization MS (ESI-MS), time-resolved electrospray ionization time-of-flight MS, Hydrogen/deuterium exchange-MS (HDX-MS) A variety of MS methods have been used to characterize Prdxs. Disulfide formation has been analyzed by ESI-MS, hyperoxidation by ESI TOF MS, and self-assemblies of Prdxs using native MS. HDX-MS relies on protein mass increases by isotopic labeling.…”

“…It was used to assess the exchange rates of hydrogen for its isotope - deuterium in the C-terminal region of oxidized Prdx suggesting the exposure of this region to solvent under oxidation. [ 138 , [142] , [143] , [144] ] Size Exclusion Chromatography (SEC) Apparent molecular weights and oligomeric properties of Prdxs are routinely analyzed by SEC. Additionally, liquid chromatography systems can be coupled to a mass spectrometer to analyze peptides.…”

Efficient Electrochemical Detection of Homocysteine in Biological Samples Based on Au NPs Multi-Walled Carbon Nanotube Composites

“…[12][13][14][15] Nevertheless, the detection of homocysteine by electrochemical methods was poorly reported. 16 Hence, the establishment of a sensitive electrochemical sensor to efficiently analyze HcySH in biological samples remained a daunting task.…”