Where Does the Electron Go? Stable and Metastable Peptide Cation Radicals Formed by Electron Transfer

Electron transfer to doubly and triply charged heptapeptide ions containing polar residues Arg, Lys, and Asp in combination with nonpolar Gly, Ala, and Pro or Leu generates stable and metastable charge-reduced ions, (M + 2H)^+●, in addition to standard electron-transfer dissociation (ETD) fragment ions. The metastable (M + 2H)^+● ions spontaneously dissociate upon resonant ejection from the linear ion trap, giving irregularly shaped peaks with offset m/z values. The fractions of stable and metastable (M + 2H)^+● ions and their mass shifts depend on the presence of Pro-4 and Leu-4 residues in the peptides, with the Pro-4 sequences giving larger fractions of the stable ions while showing smaller mass shifts for the metastables. Conversion of the Asp and C-terminal carboxyl groups to methyl esters further lowers the charge-reduced ion stability. Collisional activation and photodissociation at 355 nm of mass-selected (M + 2H)^+● results in different dissociations that give sequence specific MS³ spectra. With a single exception of charge-reduced (LKGLADR + 2H)^+●, the MS³ spectra do not produce ETD sequence fragments of the c and z type. Hence, these (M + 2H)^+● ions are covalent radicals, not ion-molecule complexes, undergoing dramatically different dissociations in the ground and excited electronic states. The increased stability of the Pro-4 containing (M + 2H)^+● ions is attributed to radicals formed by opening of the Pro ring and undergoing further stabilization by hydrogen atom migrations. UV-VIS photodissociation action spectroscopy and time-dependent density functional theory calculations are used in a case in point study of the stable (LKGPADR + 2H)^+● ion produced by ETD. In contrast to singly-reduced peptide ions, doubly reduced (M + 3H)⁺ ions are stable only when formed from the Pro-4 precursors and show all characteristics of even electron ions regarding no photon absorption at 355 nm or ion-molecule reactions, and exhibiting proton driven collision induced dissociations. Graphical Abstract ᅟ.