S-nitrosation of cysteine as evidenced by IRMPD spectroscopy

Abstract

S-nitrosation of cysteine plays an important role in storage and transport of NO, a key signaling molecule in vivo. An approach to detect this modification in the bare, charged amino acid is presented, based on IR multiple photon dissociation (IRMPD) spectroscopy. Protonated and deprotonated S-nitrosocysteine ions, [SNOCys+H]+ and [SNOCys−H]−, have been obtained by electrospray ionization and assayed for IR activity in either the 1000–1900cm−1 or the 3000–3600cm−1 wavenumber range. The so-obtained IRMPD spectra display characteristic features ascribed to the presence of the S-nitrosation motif, which are missing in the corresponding IRMPD spectra of the native cysteine ions, [Cys+H]+ and [Cys−H]−. In particular, the NO stretching mode is unambiguously identified by the red shift observed for the 15N-labelled species. The interpretation of the IRMPD spectra is supported by density functional theory calculations of the optimized geometries, relative energies and IR spectra of [SNOCys+H]+ and [SNOCys−H]−. Both sampled ions comprise a thermally averaged population of conformers contributing to the experimental IRMPD spectra. This notion is supported by the agreement between the calculated IR absorption spectra of the several conformers, and the recorded IRMPD spectrum. The gathered evidence points to a characteristic NO stretching mode that emerges as a pronounced feature at 1460–1490cm−1 in the IRMPD spectrum of [SNOCys−H]−, namely in a region where [Cys−H]− displays no IRMPD activity. Conversely, the NO stretching vibration of [SNOCys+H]+ is enclosed in a wide absorption including the CO stretching mode at 1780cm−1. The [SNOCys−H]− negative ions are thus a promising benchmark in a search for S-nitrosation features using IRMPD spectroscopy.