Identification of free radicals by spin trapping with DEPMPO and MCPIO using tandem mass spectrometry.

This study evaluates the use of a pyrroline (DEPMPO) and an imidazole (MCPIO) spin trap for the detection of hydroxyl and biomolecule (a peptide and a phospholipid) free radical adducts by Electrospray Ionization Mass Spectrometry (ESI-MS). The hydroxyl and biomolecule free radical adducts were detected using a QTOF and a linear ion trap (LIT) mass spectrometers. In the presence of hydroxyl radical, the mass spectrum obtained for each of the spin traps, DEPMPO and MCPIO, showed the presence of ions that could be attributed to hydroxyl and peroxyl radicals. Further characterisation by tandem mass spectrometry (ESI-MS/MS) revealed also the presence of hydroxy-hydroxyl adducts. Based on the results here described, we show that DEPMPO is a better spin trap for free radicals trapping and detection by mass spectrometry mainly because adducts show increased signal intensity. The ESI-MS spectra obtained for DEPMPO and MCPIO in the presence of biomolecule radicals (peptide and phospholipid) show molecular ions of DEPMPO and MCPIO adducts, which were characterised by tandem mass spectrometry. Both carbon centered radicals and oxygen centered radicals were efficiently trapped by the two spin traps and analysis of QTOF-MS/MS mass spectra allowed the location of the radical position in either the peptide or in the phospholipid fatty acyl chain. However, the tandem mass spectra of MCPIO adducts were more informative than DEPMPO adducts. The LIT-MS/MS spectra only shows typical peptide and phospholipid fragmentation, which difficult the structural characterisation of the spin adduct. In this study, the DEPMPO and MCPIO adducts were identified either in the nitrone or in the hydroxylamine form, which are ESR silent forms. The results described here show that both spin traps coupled with detection by mass spectrometry are valuable tools for trapping radicals of biomolecules. Furthermore, the acquired data provide valuable information on the presence of adducts (hydroxyl and biomolecule) that are Electron Spin Resonance (ESR) silent. This is especially important considering the complexity of the radical species in biological environment and the presence of reducing compounds that convert the spin adducts to silent ESR forms.