Hydrogen Atom Adducts to the Amide Bond. Generation and Energetics of the Amino(hydroxy)methyl Radical in the Gas Phase

118

Hydrated divalent magnesium and calcium clusters are used as nanocalorimeters to measure the internal energy deposited into size-selected clusters upon capture of a thermally generated electron. The infrared radiation emitted from the cell and vacuum chamber surfaces as well as from the heated cathode results in some activation of these clusters, but this activation is minimal. No measurable excitation due to inelastic collisions occurs with the low-energy electrons used under these conditions. Two different dissociation pathways are observed for the divalent clusters that capture an electron: loss of water molecules (Pathway I) and loss of an H atom and water molecules (Pathway II). For Ca(H 2 O) n 2ϩ , Pathway I occurs exclusively for n Ն 30 whereas Pathway II occurs exclusively for n Յ 22 with a sharp transition in the branching ratio for these two processes that occurs for n Ϸ 24. The number of water molecules lost by both pathways increases with increasing cluster size reaching a broad maximum between n ϭ 23 and 32, and then decreases for larger clusters. From the number of water molecules that are lost from the reduced cluster, the average and maximum possible internal energy is determined to be ϳ4.4 and 5.2 eV, respectively, for Ca(. This value is approximately the same as the calculated ionization energies of M(H 2 O) n ϩ , M ϭ Mg and Ca, for large n indicating that the vast majority of the recombination energy is partitioned into internal modes of the ion and that the dissociation of these ions is statistical. For smaller clusters, estimates of the dissociation energies for the loss of H and of water molecules are obtained from theory. For Mg(H 2 O) n 2ϩ , n ϭ 4 -6, the average internal energy deposition is estimated to be 4.2-4.6 eV. The maximum possible energy deposited into the n ϭ 5 cluster is Ͻ7.1 eV, which is significantly less than the calculated recombination energy for this cluster. There does not appear to be a significant trend in the internal energy deposition with cluster size whereas the recombination energy is calculated to increase significantly for clusters with fewer than 10 water molecules. These, and other results, indicate that the dissociation of these smaller clusters is nonergodic. . The effectiveness of the bottom-up method for complex samples can be enhanced by using multidimensional separations. Clemmer and coworkers elegantly demonstrated that combining on-line liquid chromatography (LC) with ion mobility spectrometry and MS can greatly improve separations without increasing analysis times over LC/MS alone [2][3][4]. In contrast, the "top-down" approach to protein characterization has the advantage that de novo sequencing, including the identification and structural localization of labile posttranslational modifications, can be done directly on protein mixtures without proteolysis [5,6]. This top-down approach has greatly benefited from the development of electron capture dissociation (ECD), a method pioneered by McLafferty and coworkers [6][7][8][9]. In a typical ECD experim...

Section: Internal Energy Depositionmentioning

confidence: 99%

Nonergodicity in electron capture dissociation investigated using hydrated ion nanocalorimetry

Leib

Donald

Bush

et al. 2007

118

“…First, what factors influence the formation of the unstable amino ketyl radical that is believed to be an intermediate for the N À C a bond cleavage [14]? The formation of this intermediate is lively debated, especially the factors concerning the location of the initial electron attachment to peptide cations [15][16][17][18][19][20]. The second critical step encompasses the fragmentation reactions occurring after formation of the amino ketyl radical, such as the losses of atomic hydrogen, side chains [21] or N À C a bond cleavage via primary of secondary [22,23] radical reactions.…”

Section: Introductionmentioning

confidence: 99%

“…The second critical step encompasses the fragmentation reactions occurring after formation of the amino ketyl radical, such as the losses of atomic hydrogen, side chains [21] or N À C a bond cleavage via primary of secondary [22,23] radical reactions. These fragmentation pathways are believed to be largely determined by the energy levels of the formed products as well as their transition states [15,16].…”

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.

“…Both the hydrogen atom elimination and migration have been shown to be very fast reactions in thermal peptide radicals, and the branching ratios for these two channels are affected by tunneling effects [13]. Recapture of a hydrogen atom by an amide group must overcome an energy barrier on the order of 50 kJ mol Ϫ1 and can occur at the oxygen or carbon atoms in neutral amides [10]. The aminoketyl radicals formed by hydrogen transfer to the amide carbonyl oxygen are weakly bound with respect to NOC ␣ bond cleavage, which is thermoneutral or exothermic in peptides and requires only a 30 -50 kJ mol Ϫ1 energy barrier [14,15].…”

mentioning

confidence: 99%

“…A fundamental problem of the hot hydrogen atom mechanism is that an H atom addition to an amide carbonyl group is expected to occur at both oxygen and carbon atoms for which high-level ab initio calculations predict very similar activation energies [10,11]. The Scheme 1.…”

mentioning

confidence: 99%

Toward a general mechanism of electron capture dissociation

Syrstad

Turecček

2005

Self Cite

309

358

The effects of positive charge on the properties of ammonium and amide radicals were investigated by ab initio and density functional theory calculations with the goal of elucidating the energetics of electron capture dissociation (ECD) of multiply charged peptide ions. The electronic properties of the amide group in N-methylacetamide (NMA) are greatly affected by the presence of a remote charge in the form of a point charge, methylammonium, or guanidinium cations. The common effect of the remote charge is an increase of the electron affinity of the amide group, resulting in exothermic electron capture. The NOC ␣ bond dissociation and transition state energies in charge-stabilized NMA anions are 20 -50 kJ mol Ϫ1 greater than in the hydrogen atom adduct. The zwitterions formed by electron capture have proton affinities that were calculated as 1030 -1350 kJ mol Ϫ1, and are sufficiently basic for the amide carbonyl to exothermically abstract a proton from the ammonium, guanidinium and imidazolium groups in protonated lysine, arginine, and histidine residues, respectively. A new mechanism is proposed for ECD of multiply charged peptide and protein cations in which the electron enters a charge-stabilized electronic state delocalized over the amide group, which is a superbase that abstracts a proton from a sterically proximate amino acid residue to form a labile aminoketyl radical that dissociates by NOC ␣ bond cleavage. This mechanism explains the low selectivity of NOC ␣ bond dissociations induced by electron capture, and is applicable to dissociations of peptide ions in which the charge carriers are metal ions or quaternary ammonium groups. The new amide superbase and the previously proposed mechanisms of ECD can be uniformly viewed as being triggered by intramolecular proton transfer in charge-reduced amide cation-radicals. In contrast, remote charge affects NOH bond dissociation in weakly bound ground electronic states of hypervalent ammonium radicals, as represented by methylammonium, CH 3 NH 3 · , but has a negligible effect on the NOH bond dissociation in the strongly bound excited electronic states. This refutes previous speculations that loss of "hot hydrogen" can occur from an excited state of an ammonium radical. , result in backbone dissociations yielding series of sequence-specific ions. In addition to dissociations of closed-shell cations, peptide and protein cation-radicals have recently been shown to undergo different types of dissociations that are often complementary to those of closed shell cations, and thus represent a useful tool for protein sequencing [4, 5]. The most general method of producing peptide cation-radicals in the gas phase is by exposing multiply protonated peptides to low-energy electrons, which leads to exothermic ion-electron recombination [6]. The resulting peptide cation-radicals dissociate by a variety of reactions, namely, loss of H atoms, ammonia, and side chain fragments, cleavage of disulfide bonds, and, in particular, backbone cleavages. This electron capture dissociation...