Probing the Mechanism of Electron Capture and Electron Transfer Dissociation Using Tags with Variable Electron Affinity

Abstract

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) of doubly protonated electron affinity (EA)-tuned peptides were studied to further illuminate the mechanism of these processes. The model peptide FQpSEEQQQTEDELQDK, containing a phosphoserine residue, was converted to EA-tuned peptides via β-elimination and Michael addition of various thiol compounds. These include propanyl, benzyl, 4-cyanobenzyl, perfluorobenzyl, 3,5-dicyanobenzyl, 3-nitrobenzyl and 3,5-dinitrobenzyl structural moieties, having a range of EA from -1.15 to 1.65 eV, excluding the propanyl group. Typical ECD or ETD backbone fragmentations are completely inhibited in peptides with substituent tags having EA over 1.00 eV, which are referred to as electron predators in this work. Nearly identical rates of electron capture by the dications substituted by the benzyl (EA = -1.15 eV) and 3-nitrobenzyl (EA = 1.00 eV) moieties are observed, which indicates the similarity of electron capture cross sections for the two derivatized peptides. This observation leads to the inference that electron capture kinetics are governed by the long range electron-dication interaction and are not affected by side chain derivatives with positive EA. Once an electron is captured to high-n Rydberg states, however, through-space or through-bond electron transfer to the EA-tuning tags or low-n Rydberg states via potential curve crossing occurs in competition with transfer to the amide π* orbital. The energetics of these processes are evaluated using time-dependent density functional theory with a series of reduced model systems. The intramolecular electron transfer process is modulated by structure-dependent hydrogen bonds and is heavily affected by the presence and type of electron withdrawing groups in the EA-tuning tag. The anion radicals formed by electron predators have high proton affinities (approximately 1400 kJ/mol for the 3-nitrobenzyl anion radical) in comparison to other basic sites in the model peptide dication, facilitating exothermic proton transfer from one of the two sites of protonation. This interrupts the normal sequence of events in ECD or ETD leading to backbone fragmentation by forming a stable radical intermediate. The implications which these results have for previously proposed ECD and ETD mechanisms are discussed.

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].…”

“…In the former case, the inhibition of backbone bond cleavages was explained by the electron predator mechanism [36]. In the latter case, the authors suggested that hydrogen bonding between the carbonyl oxygen of the glutamic acids side chains and the backbone amide hydrogen results in stabilization of the charge-reduced species with subsequent inhibition of the ECD fragmentation [38].…”

Effects of Peptide Backbone Amide-to-Ester Bond Substitution on the Cleavage Frequency in Electron Capture Dissociation and Collision-Activated Dissociation

“…Thus, the anion formed by the ester carbonyl requires less Coulomb stabilization than the OCN π* orbital. However, Beauchamp and co-workers [32] showed that electron capture and cleavage of amide bonds were only affected when substituent tags in the peptide had EA greater than ca. 50 kJ/mol.…”

“…Turecek and co-workers prevented ECD backbone cleavages by the addition of a stable electron and hydrogen atom trap to peptide ions [31]. More recently, Beauchamp and co-workers inserted tags with variable electron affinities (EA; -1.15 to +1.65 eV) into a peptide sequence and analyzed the model peptides by ECD and ETD [32]. Two main conclusions were drawn.…”