2-Methyl-2-nitrosopropane, a promising label for biological thiol targets: a combined EPR and ESI-MS/MS study.

摘要

Nitroxide radicals are stable paramagnetic species presenting a wide range of applications throughout material science, chemistry and biochemistry. In particular, site-directed spin labeling, a technique in which a nitroxide probe is covalently linked to the thiol group of a cysteinyl residue, has emerged as a powerful method for investigating protein structure, dynamics and conformational changes [1, 2]. We have previously shown that the addition of reduced glutathione, a tripeptide containing one cysteinyl residue, on 2-methyl-2-nitrosopropane (MNP) occurred in a fast process in oxidative media to generate spin adducts [3]. Since this interesting reactivity could open a new route to spin-label proteins, the influence of thiol structure on the stability of the nitroxides formed upon their reaction with MNP was scrutinized. Electrospray tandem mass spectrometry (ESI-MS/MS) has been employed in conjunction with electronic paramagnetic resonance (EPR) to characterize nitroxides and their diamagnetic derivatives, even in complex matrices [4, 5]. EPR is generally the technique of choice to study nitroxides owing to their paramagnetic nature, the spectra recorded being very sensitive to their mobility and to their environment. However, only poor structural information can be gained by EPR, nitroxides with similar structures generally yielding identical spectra. As a result, very useful data were extracted from collision induced dissociation spectra of protonated species derived from nitroxides, such as hydroxylamine, nitrone or alkoxylamine.