Generation of hydrogen radicals for reactivity studies in Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) studies of two modified amyloid {3 peptides (20-29 and 25-35) were performed to investigate the role of H" radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H" generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z" ions were still observed in their ECD spectra. Thus, although H" transfer may be important for the N--C", bond cleavage, there also exist other pathways, which would require a radical migration via H" abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides. aAm

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

The effect of fixed charge modifications on electron capture dissociation

Cournoyer

Lin

et al. 2008

“…This model is often referred as a "hot hydrogen atom" mechanism [24]. Since the cross-section for direct capture of free hydrogen atoms by gas-phase peptides is quite low [25] , it appears that mainly hydrogen atoms from neutralized protonated groups solvated to carbonyls can be effectively transferred to carbonyl oxygens [23]. An alternative mechanism was put forward independently by Turecek and Simons and coworkers [26][27][28].…”

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.

“…One problem is that the cross sections for hydrogen atom capture by gas-phase peptide ions are low, as evidenced by the absence of addition or dissociation reactions when peptide ions were exposed to H atoms in an ICR cell [21]. This is corroborated by ab initio studies that showed that H atom additions to amide carbonyl groups have substantial energy barriers and are expected to be negligibly slow in thermal species for which transition state theory calculations give rate constants on the order of 10 Ϫ18 molecule cm Ϫ3 s Ϫ1 [13].…”

mentioning

confidence: 99%

Toward a general mechanism of electron capture dissociation

Syrstad

Turecček

2005

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...