Collision-induced dissociations of protonated O-18-labeled tetraglycines labeled separately at either the first or the second amide bond established that water loss from the backbone occurs from the N-terminal residue. Density functional theory at B3LYP/6-311++G(d,p) predicted that the low-energy [G(4) + H - H2O](+) product ion is an NI-protonated 3,5-dihydro-4H-imidazol-4-one. The ion at the lowest energy, III, is 24.8 kcal mol(-1) lower than the protonated oxazole structure, II, proposed by Bythell et al.(J. Phys. Chem A 2010, 114, 5076-5082). In addition, structure III has a predicted IR spectrum that provides a better match with the published experimental IRMPD spectrum than that of structure II.