Peptide cation‐radicals. A computational study of the competition between peptide NCα bond cleavage and loss of the side chain in the [GlyPhe‐NH2 + 2H]+• cation‐radical

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: 78%

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confidence: 99%

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confidence: 99%

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

Jones

Sasaki

Goodlett

et al. 2007

“…Because gramicidin S is cyclic, these backbone fragments require two cleavages from a single electroncapture event and are thought to arise from a freeradical cascade [28]. While it is pertinent to contemplate whether the reduced number of fragments is attributable to the radical cascade terminating earlier at the lower internal energies at 86 K, considering the low activation barriers of these gas-phase radical reactions [18,27] it is not sufficient without invoking a conformational argument. As with the Substance P results, the smaller number of fragments is consistent with a smaller conformer population and reduced thermal fluctuations.…”

Section: Resultsmentioning

confidence: 99%

Electron capture dissociation at low temperatures reveals selective dissociations

Mihalca

Kleinnijenhuis

McDonnell

et al. 2004

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve N™C ␣ cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population. ( ploys reactions of multiprotonated peptides/ proteins with low energy electrons to generate peptide fragments. The high sequence coverage and the ability to retain labile groups after ECD have allowed posttranslational modifications (PTMs) and point mutations (PMs) in peptides and proteins to be both identified and localized. Such performance is of great value for the analysis of the proteome, particularly that of diseased organisms. PTMs and PMs are widespread and are frequently associated with disease [5][6][7], for example over 80 different point mutations have been found in transthyretin (most of which lead to autosomal disorders) [8].To be able to fully characterize such modified proteins, 100% sequence coverage is required (so-called top-down proteomics). Several methods have been developed to increase the sequence coverage of ECD, which can be grouped into either infrared illumination [9 -11] or collisional activation [12]. These methods increase the average internal energy of the ions, which also affects the conformation of these gas-phase ions [13].There is experimental and theoretical evidence that ECD is directed by the internal solvation of the (neutralized) proton [9, 14 -18]. If the reaction progresses faster than the electron-proton recombination energy is randomized and thermal fluctuations are small, specific fragments would be expected from a single (frozen) conformer, reflecting the solvation shell, specific for that conformation, surrounding the neutralized proton. Thermal fluctuations that result in a dynamic solvation shell, but not conformational change (local fluctuations rather than a global change), would produce a greater number of fragments. Finally, multiple conformations as well as thermal fluctuations would produce yet more fragments. Because the methods used to increase the sequence coverage of ECD all increase the internal energy of the ions, and so affect the conformation of the gas-phase ions, the...

“…ECD is complementary to traditional MS/MS methods, such as CAD, in that NOC ␣ and SOS bonds are cleaved, the former resulting in c=-and z · ions [31,32], without loss of labile PTMs [27][28][29][30]. Although ECD has been successfully applied to analyze intact proteins [33,34], its detailed mechanism is still an active controversy for both experimentalists and theoretists [27,[35][36][37][38][39][40][41][42][43][44][45][46]. In one of the proposed peptide ECD mechanisms [29,36,41], neutralization is thought to occur at a charge center (H ϩ ) (solvated to, e.g., carbonyl groups), which releases a hydrogen atom that is subsequently captured by a backbone carbonyl group, forming an aminoketyl intermediate.…”

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confidence: 99%

Divalent metal ion-peptide interactions probed by electron capture dissociation of trications

Liu

Håkansson

2006

Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP ϩ H ϩ M] 3ϩ (M 2ϩ ϭ Mg 2ϩ -Ba 2ϩ and Mn 2ϩ -Zn 2ϩ ) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c= and z · -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y=-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (c n =M Ϫ H) ϩ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful