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 quadrupole time-of-flight (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 (MS/MS) also revealed the presence of hydroxy–hydroxyl adducts. Based on the results described here, we show that DEPMPO is a better spin trap for free radical 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 MS/MS. Both carbon centred radicals and oxygen centred radicals were efficiently trapped by the two spin traps and analysis of QToF-MS/MS spectra allowed the location of the radical position in either the peptide or in the phospholipid fatty acyl chain. However, the MS/MS spectra of MCPIO adducts were more informative than DEPMPO adducts. The LIT-MS/MS spectra only shows typical peptide and phospholipid fragmentation, which is difficult for the structural characterisation of the spin adduct. In this study, the DEPMPO and MCPIO adducts were identified either in nitrone or hydroxylamine form, which are electron spin resonance (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 ESR silent. This is especially important considering the complexity of the radical species in a biological environment and the presence of reducing compounds that convert the spin adducts to silent ESR forms.
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