Low energy CID and action IRMPD provide insights into a minor subpopulation of the gas-phase conformers of triply charged bradykinin

Abstract

Unique fragmentation of the IMS-resolved conformers of triply charged bradykinin is shown to occur at very low collision energies by quadrupole CID. For the major C conformation of bradykinin, the unique fragmentation appears to result from a subpopulation that has a different average CCS than the dominant C population that has been previously identified as TTC (trans, trans, cis at the three Pro residues). The three major gas-phase isomers exhibit minimal differences in fragmentation at collision energies greater than 12eV, presumably because the amide bonds of the proline residues isomerize prior to dissociation at these higher energies. The differences in fragmentation observed in the low collision energy regime (<12eV) are attributed to the conformational differences of the fragmenting subpopulations of the mobility resolved populations associated with the cis or trans isomerization state of the three proline residues. Action IRMPD of the b2 ion supports a cis-Pro2 isomerization state for the subpopulation of conformation C fragmenting at low energies, assuming no isomerization was induced by the low energy CID. Substitution of N-methyl alanine, an acyclic proline mimic, independently, for each of the three prolines of bradykinin and low energy fragmentation of the minor subpopulation of conformation C allowed for tentative assignments of the isomerization state of two of the three proline resides of this subpopulation. Density functional theory calculations support the conclusion made from NMA-substitutions and together suggest that the subpopulation of conformation C is cis at Pro2 and trans at Pro7. It is not clear whether the minor subpopulation is a typical feature of triply charged bradykinin or whether unintended activation in the instrument used here produces this population. The results illustrate the utility of ion mobility coupled with low-energy CID for the determination of a minor subpopulation that was not visible by MS or IM alone.