Electron transfer dissociation (ETD) of doubly and triply charged ions from the amphipathic N-terminal decapeptide GFLSILKKVL-NH2 segment of melectin gave different distributions of fragment ions. The triply charged ions generated extensive series of fragment ions of c and z type that covered the entire sequence from both the N and C termini. In contrast, electron transfer to the doubly charged ions caused backbone cleavages that occurred at residues close to the N and C termini. Attachment of a free low-energy electron to the doubly charged ions caused primary dissociations close to the N and C termini that were followed by consecutive dissociations of z ions. The structure of gaseous doubly charged ions from the melectin peptide was elucidated by a combination of exhaustive conformational search by force-field molecular dynamics, large-scale gradient optimization using the semiempirical PM6 method, and density functional theory single-point energy and gradient optimization calculations. The most stable doubly charged ions were found to be protonated at the lysine ε-amino groups and have globular conformations. The backbone cleavages in ETD correlated with the electronic structure of cation-radicals produced by electron attachment to the most stable conformers. The charged lysine ammonium groups direct the incoming electron to the π* orbitals at the proximate amide groups at Phe, Leu, Lys and Val residues that show the highest spin densities. Electron attachment at these amide groups weakens the N–Cα bonds between the Phe-Leu, Leu-Ser, Lys-Lys and Lys-Val residues and causes backbone dissociations.
Electron transfer dissociation with supplemental activation to differentiate aspartic and isoaspartic residues in doubly charged peptide cations
