The effect of radical trap moieties on electron capture dissociation spectra of substance P

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 ...

Section: Sociated (Bpy ϩ H)supporting

confidence: 79%

Section: Sociated (Bpy ϩ H)mentioning

confidence: 68%

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

Jones

Sasaki

Goodlett

et al. 2007

“…O'Connor and co-workers have studied the role of radicals in ECD by chemically adding to peptides a variety of tags which act as free radical traps [29,30]. In those studies, the addition of one or two radical traps to doubly charged peptides significantly reduced or blocked the ECD backbone cleavage channels.…”

Section: Introductionmentioning

confidence: 99%

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

Kjeldsen

Zubarev

2011

Probing the mechanism of electron capture dissociation on variously modified model peptide polycations has resulted in discovering many ways to prevent or reduce N À C ! bond fragmentation. Here we report on a rare finding of how to increase the backbone bond dissociation rate. In a number of model peptides, amide-to-ester backbone bond substitution increased the frequency of O À C ! bond cleavage (an analogue of N À C ! bonds in normal peptides) by several times, at the expense of reduced frequency of cleavages of the neighboring N À C ! bonds. In contrast, the ester linkage was only marginally broken in collisional dissociation. These results further highlight the complementarity of the reaction mechanisms in electron capture dissociation (ECD) and collision-activated dissociation (CAD). It is proposed that the effects of amide-to-ester bond substitution on fragmentation are mainly due to the differences in product ion stability (ECD, CAD) as well as proton affinity (CAD). This proposal is substantiated by calculations using density functional theory. The implications of these results in relation to the current understanding of the mechanisms of electron capture dissociation and electron transfer dissociation are discussed.

“…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.…”

mentioning

confidence: 99%

Natural structural motifs that suppress peptide ion fragmentation after electron capture

Chan

2010

Series of doubly and triply protonated diarginated peptide molecules with different number of glutamic acid (E) and asparagine (N) residues were analyzed under ECD conditions. ECD spectra of doubly-protonated peptides show a strong dependence on the number of E and N residues. Both the backbone cleavages and hydrogen radical (H • ) loss from the charge-reduced precursor ions ([Mϩ2H] ϩ• ) were suppressed as the number of E and N residues increases. A strong inhibition of the backbone cleavages and H • loss from [Mϩ2H] ϩ• was found for peptides with 6E residues (or 4E ϩ 2N residues). The results obtained using these model peptides were re-confirmed by analyzing N-arginated Fibrinopeptide-B (i.e., REGVNDNEEGFFSAR). In contrast to the N-arginated peptide, ECD of the doubly-protonated Fibrinopeptide-B and its analogues show extensive backbone cleavages leading to series of c-and z-ions (ϳ80% sequence coverage). Based on these results, it is believed that peptide ions with all surplus protons sequestered in arginine-residues would show enhanced stability under ECD conditions as the number of acid-residue increases. The suppression of backbone cleavages and H • loss from [Mϩ2H] ϩ• are presumably attributed to the low reactivity of the charge-reduced precursor ions. One of the possible hypothesis is that diarginated E-rich peptides may contain hydrogen bonds between carbonyl oxygen of E side chains and backbone amide hydrogen. These hydrogen bonds would provide extra stabilization for [Mϩ2H] -4]. ECD also plays a useful role in characterization and localization of post-translational modifications (PTMs) as it preserves labile PTMs in protein [5][6][7] while cleaving the protein backbone to give series of specific fragment ions. This is in contrast with that of conventional slow-heating ion dissociation methods [8,9], such as collision induced dissociation (CID) [10 -12] and infrared multiproton dissociation (IRMPD) [13]. ECD involves the reaction between multiply-charged ions and low-energy electrons. Besides the formation of the charge-reduced precursor ions, [M ϩ nH] (n-1)ϩ• , the recombination energy released might provide enough energy to cause backbone N-C ␣ cleavages producing series of c or z • ions and to a lesser extent series of a • or y ions. Other ECD events include the elimination of (1) a H • to form [M ϩ (n-1)H] (n-1)ϩ , and (2) amino acid side chains from z • ions and/or [M ϩ nH] (n-1)ϩ• . Cleavages in ECD are nonspecific [14], and the relative propensities for dissociation of various amino acid residues were found to fall in a narrow range. Because of the ring-type structure, only fragment ions resulting from the backbone cleavages at the N-terminal side of proline (P) were suppressed [15].The importance of radical in charge-reduced precursor ion in ECD was investigated by synthetically incorporating single and multiple radical trap, spin trap, and charge tag moieties in model peptides. In radical trap experiments, Belyayev et al. [16] attached coumarin labels onto the N-terminal amino group (or/an...