Electron capture in spin-trap capped peptides. An experimental example of ergodic dissociation in peptide cation-radicals

Abstract

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4=-carboxypyrid-2=-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH 2 , pepy-AQEFGEQGQKALKQL-NH 2 , and pepy-AQEGSE-QAQKFFKQL-NH 2 . Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b 11-14 ϩ 1) ϩ· fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1= positions in the pepy moiety had topical gas-phase basicities (GB ϭ 923 kJ mol Ϫ1 ) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE ϭ 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by Ͼ160 kJ mol Ϫ1 , which exceeded the hydrogen atom affinity of the backbone amide groups (21-41 kJ mol Ϫ1 ). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD. . Mechanistic details of peptide cation dissociations and the product ion structures have been elucidated by quantum chemical calculations [2][3][4] and appear to provide a coherent model of the peptide gas-phase ion chemistry [5]. Inherent to the mechanistic model of peptide ion fragmentations is the concept of efficient internal vibrational energy redistribution (IVR) in the dissociating peptide ion [6], so that the dissociation can be treated as an ergodic process by appropriate kinetic schemes, such as the RiceRamsperger-Kassel-Marcus [7] or transition-state theories [8]. The concept of IVR has received experimental support by time-resolved photodissociation studies [9], and is consistent with the mode of excitation upon collisional activation at low kinetic energies (Ͻ100 eV) or upon absorption of infrared photons [10].The current understanding is less definite concerning the dissociations of peptide cation-radicals produced by electron capture or transfer [11]. In electron ...

Natural structural motifs that suppress peptide ion fragmentation after electron capture

“…In radical trap experiments, Belyayev et al [16] attached coumarin labels onto the N-terminal amino group (or/and lysine side chain) and demonstrated that the presence of a radical trap in a peptide ion could inhibit backbone fragmentation under typical ECD conditions. Similar observations were also made by Jones and coworkers using 2-(4=-carboxypyrid-2=-yl)-4-carboxamide group (pepy) [17] as spin trap label. In fixed charge experiments, Li et al attached 2,4,6-trimethylpyridinium (TMP) [18] to the N-terminal amino group (or/and lysine side chain) of some model peptides and showed that the usual backbone cleavage fragments were suppressed and some label-related neutral losses from the reduced precursor ions were found.…”

Electron Capture Dissociation of Hydrogen-Deficient Peptide Radical Cations

“…The suppressed backbone bond cleavages observed following ECD of the hydrogen-deficient species is analogous to the results obtained in ECD of peptides modified with tags having high electron affinity [36], peptides derivatized with fixed charge tags [34,35], radical and hydrogen atom traps [35], nitrated peptides [37], and peptides featuring a high number of glutamic acid and asparagine residues [38]. These reports [35][36][37][38] and our results highlight the importance of the reactive aminoketyl intermediate in ECD and indicate that N-C α bond cleavages are largely quenched, or eliminated, when the formation of the reactive aminoketyl radical intermediate is inhibited.…”

“…ECD on peptides carrying fixed charge tags [34,35] and electron traps [36] has been performed to examine the effect of these groups on the dissociation outcome. For instance, 2,4,6-trimethylpyridinium (TMP), which was postulated to act as a radical trap and also inhibits hydrogen atom formation, was used as a fixed charge tag for two amyloid β peptides [34].…”

Formation of Peptide Radical Cations (M+·) in Electron Capture Dissociation of Peptides Adducted with Group IIB Metal Ions

“…The anionic radical formed at the carbonyl group is even more basic than the side chain of the arginine residue and would induce abstraction of the nearby proton. The ketylamino radical formed would undergo cleavage along the N-C α linkage to form the usual c-/z-ions [13][14][15][16][17][18][19].…”