Peptide fragmentation can lead to an oxazolone or diketopiperazine b2+ ion structure. IRMPD spectroscopy combined with computational modeling and gas-phase H/D exchange was used to study the structure of the b2+ ion from protonated HAAAA. The experimental spectrum of the b2+ ion matches both the experimental spectrum for the protonated cyclic dipeptide HA (a commercial diketopiperazine) and the theoretical spectrum for a diketopiperazine protonated at the imidazole pi nitrogen. A characteristic band at 1875 cm−1, and increased abundance of the peaks at 1619 and 1683 cm−1, indicate a second population corresponding to an oxazolone species. H/D exchange also shows a mixture of two populations consistent with a mixture of b2+ ion diketopiperazine and oxazolone structures.

Infrared multiple photon dissociation (IRMPD) spectroscopy combined with theoretical vibrational spectra provides a powerful tool for probing structure. This technique has been used to probe the structure of protonated cyclic AG and the b2+ ion from AGG. The experimental spectrum for protonated cyclo AG compares very well with the theoretical spectra for a diketopiperazine. The structure is most likely a combination of protonation at the alanine and glycine amide oxygens. The experimental spectrum for the b2+ ion from protonated AGG matches best to the theoretical spectrum for an oxazolone structure protonated on the ring nitrogen. In particular, the carbonyl stretching band at 1970 cm-1 is blue-shifted by about 200 cm-1 compared to the experimental spectrum for protonated cAG, indicating that these two structures are distinct. This is the first time that a direct IR spectrum of a b2+ ion has been obtained and, for this ion, the oxazolone structure proposed based on prior calculations and experiments is confirmed by a spectroscopic method.