Novel identification of a sulfur-centered, radical-derived 5,5-dimethyl-1-pyrroline N-oxide nitrone adduct formed from the oxidation of DTT by LC/ELISA, LC/electrospray ionization-MS, and LC/tandem MS

The detection of highly reactive free radicals generated in biological systems by an ESR spin-trapping technique is always difficult and limited due to the short lifetimes of ESR active spin-trapping radical adducts and poor structural information provided by ESR spectra. In this investigation, we have for the first time employed anti-5,5-dimethyl-1-pyrroline N-oxide (DMPO) polyclonal antiserum that specifically recognizes stable, ESR silent end products of DMPO radical adducts and combined HPLC with ELISA, electrospray ionization mass spectrometry (ESI-MS), and tandem mass spectrometry (MS/MS) to separate and characterize DMPO nitrone adducts derived from free radical metabolites. When mireoperoxidase-11 (MP-11) reacted with DTT in the presence of DMPO with or without H2O2, we detected radical-derived DMPO nitrone adducts by ELISA. Similar results were obtained when MP-11 was replaced by hemin. To identify the DMPO nitrone adducts formed in both reaction systems, LC separation was carried out, and the fractions eluted from the LC column were collected and analyzed by ELISA. In both reaction mixtures, we found that only one peak with the same retention time showed a strong positive ELISA signal, suggesting that this peak was from radical-derived DMPO nitrone adducts and that both systems produced the same free radical metabolites. Using online LC/ESI-MS, LC/MS/MS, and H-1 NMR, we demonstrated that the DMPO nitrone adducts formed are from the DMPO adducts of the sulfur-centered radical of DTT. The successful application of LC/ELISA, LC/MS, and LC/MS/MS in this study makes it possible to separate and identify the stable DMPO nitrone adducts derived from free radical metabolites generated in biological systems.