Gas-phase ions of protonated l-glutathione as native species, [GSH + H](+), and S-nitroso derivative, [GSNO + H](+), have been generated by electrospray ionization and probed via infrared multiple photon dissociation (IRMPD) action spectroscopy. Insight into the conformational landscape is gained from interpretation of the IR spectra aided by high-level theoretical calculations, which enables structural assignment disclosing both the site of protonation and the intramolecular hydrogen-bond network. Calculations yield the low-energy structures of [GSNO + H](+). A admixture of the four most stable ones (SN1, AN1, SN2, and AN2) is apt to account for the experimental IRMPD spectra obtained in both the 10002000 and the 31003700 cm(1) spectral ranges. The most stable form of [GSNO + H](+), SN1, protonated at the amino group, presents a syn conformation at the SN (partial) double bond and all peptidic carbonyls involved in (strong) C=O center dot center dot center dot HN hydrogen bonds, so allowing closure of a C5 (beta-strand), two C7 (gamma-turn), and one C9-membered rings. An appreciable barrier to rotation of 43 kJ mol(1) about the SN bond is found to separate SN1 from the analogous anti isomer AN1, which lies only 0.70 kJ mol(1) higher in free energy. Conformers obtained for [GSH + H](+) are very similar to the [GSNO + H](+) counterparts, indicating that the S-nitrosation motif does not affect significantly the geometry of the peptide. The observed nu(NO) signatures at 1622 and 1690 cm(1), merged with other absorptions, are revealed by their sensitivity to (NO)-N-15 isotope labeling and by comparison with the IRMPD spectrum of native [GSH + H](+), providing a diagnostic probe for the S-nitrosation feature in natural peptides.