Charge-dependent dissociation of insulin cations via ion/ion electron transfer

The dissociation reactions of various charge states of insulin cations obtained directly from nano-electrospray were investigated as a result of ion/ion electron transfer from azobenzene anions. Data were collected with and without simultaneous ion trap collisional excitation of the first generation charge-reduced product during the ion/ion reaction period. Neither separation of the two constituent chains nor cleavages within the loop defined by the disulfide bridges were observed under normal electron transfer dissociation (ETD) conditions for any of the charge states studied. However, substantial sequence coverage (exocyclic region: 82.6%; entire protein: 38.8%) outside the ring structure was obtained for insulin +6, while only limited coverage (exocyclic: 43.5%; entire protein: 20.4%) was observed for insulin +5 and no dissociation, aside from low abundance side-chain losses, was noted for insulin +4 and +3 in the normal ETD spectra. When the first generation charge-reduced precursor ions were subjected to collisional activation during the ion/ion reaction period, higher sequence coverages were obtained for both insulin +5 (entire protein: 34.7%) and +4 (entire protein: 20.4%) with backbone cleavages occurring within the loop defined by the disulfide bonds. Dissociation of insulin +3 was not significantly improved by the additional activation. Separation of the two constituent chains resulting from cleavages of both of the two disulfide bridges that link the chains was observed for insulin +6, +5, and +4 when the charge-reduced species were activated. The dissociation of disulfide linkages in this study suggests that as the charge state decreases, disulfide bond cleavages dominate over N–Cα bond cleavages in the electron transfer dissociation process.