Electron Capture in Charge-Tagged Peptides. Evidence for the Role of Excited Electronic States

Electron capture dissociation (ECD) was studied with doubly charged dipeptide ions that were tagged with fixed-charge tris-(2,4,6-trimethoxyphenyl)phosphonium-methylenecarboxamido (TMPP-ac) groups. Dipeptides GK, KG, AK, KA, and GR were each selectively tagged with one TMPP-ac group at the N-terminal amino group while the other charge was introduced by protonation at the lysine or arginine side-chain groups to give (TMPP-ac-peptide + H)²⁺ ions by electrospray ionization. Doubly tagged peptide derivatives were also prepared from GK, KG, AK, and KA in which the fixed-charge TMPP-ac groups were attached to the N-terminal and lysine side-chain amino groups to give (TMPP-ac-peptide-ac-TMPP)²⁺ dications by electrospray. ECD of (TMPP-ac-peptide + H)²⁺ resulted in 72% to 84% conversion to singly charged dissociation products while no intact charge-reduced (TMPP-ac-dipeptide + H)^{+{bullet operator}} ions were detected. The dissociations involved loss of H, formation of (TMPP + H)⁺, and N-C_α bond cleavages giving TMPP-CH₂CONH₂⁺ (c₀) and c₁ fragments. In contrast, ECD of (TMPP-ac-peptide-ac-TMPP)²⁺ resulted in 31% to 40% conversion to dissociation products due to loss of neutral TMPP molecules and 2,4,6-trimethoxyphenyl radicals. No peptide backbone cleavages were observed for the doubly tagged peptide ions. Ab initio and density functional theory calculations for (Ph₃P-ac-GK + H)²⁺ and (H₃P-ac-GK + H)²⁺ analogs indicated that the doubly charged ions contained the lysine side-chain NH₃⁺ group internally solvated by the COOH group. The distance between the charge-carrying phosphonium and ammonium atoms was calculated to be 13.1-13.2 Å in the most stable dication conformers. The intrinsic recombination energies of the TMPP⁺-ac and (GK + H)⁺ moieties, 2.7 and 3.15 eV, respectively, indicated that upon electron capture the ground electronic states of the (TMPP-ac-peptide + H)^{+{bullet operator}} ions retained the charge in the TMPP group. Ground electronic state (TMPP-ac-GK + H)^{+{bullet operator}} ions were calculated to spontaneously isomerize by lysine H-atom transfer to the COOH group to form dihydroxycarbinyl radical intermediates with the retention of the charged TMPP group. These can trigger cleavages of the adjacent N-C_α bonds to give rise to the c₁ fragment ions. However, the calculated transition-state energies for GK and GGK models suggested that the ground-state potential energy surface was not favorable for the formation of the abundant c₀ fragment ions. This pointed to the involvement of excited electronic states according to the Utah-Washington mechanism of ECD.