Electron transfer dissociation of photolabeled peptides. Backbone cleavages compete with diazirine ring rearrangements.

Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GLGGK  +  2H)(2+) and (GLGGK-NH2 + H)(+) ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GLGGK  +  2H)(2+), (GLGGK-NH2 + 2H)(2+),(GLGGKK + 2H)(2+), (GLGLK + 2H)(2+), and (GLLGK + 2H)(2+) showed 16 %-21 % fragment ions originating by radical rearrangements and cleavages in the diazirine ring. These side-chain dissociations resulted in eliminations of N2H3, N2H4, [N2H5], and [NH4O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion-molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the diazirine and amide π electronic systems in the low electronic states of the cation-radicals. The diazirine moiety in GL*GGK-NH2was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.