Top-Down Charge Transfer Dissociation (CTD) of Gas-Phase Insulin: Evidence of a One-Step,Two-Electron Oxidation Mechanism

Top-down analyses of protonated insulin cations of charge states of 4+, 5+, or 6+ were performed by exposing the isolated precursor ions to a beam of helium cations with kinetic energy of more than 6 keV, in a technique termed charge transfer dissociation (CTD). The ~100 ms charge transfer reaction resulted in approximately 20% conversion efficiency to other intact charge exchange products (CTnoD), and a range of low abundance fragment ions. To increase backbone and sulfide cleavages, and to provide better structural information than straightforward MS² CTD, the CTnoD oxidized products were isolated and subjected to collisional activation at the MS³ level. The MS³ CTD/CID reaction effectively broke the disulfide linkages, separated the two chains, and yielded more structurally informative fragment ions within the inter-chain cyclic region. CTD also provided doubly oxidized intact product ions at the MS² level, and resonance ejection of the singly oxidized product ion revealed that the doubly oxidized product originates directly from the isolated precursor ion and not from consecutive CTD reactions of a singly oxidized intermediate. MS⁴ experiments were employed to help identify potential radical cations and diradical cations, but the results were negative or inconclusive. Nonetheless, the two-electron oxidation process is a demonstration of the very large potential energy (>20 eV) available through CTD, and is a notable capability for a 3D ion trap platform.

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