Non-dissociative Electron Transfer Research Articles

Radical-Pairing Interactions in a Molecular Switch Evidenced by Ion Mobility Spectrometry and Infrared Ion Spectroscopy.

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch‐based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non‐dissociative electron transfer as a reductive stimulus in the gas phase and probe the consecutive co‐conformational transitions of a donor‐acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. A comparison of collision cross section distributions for analogous closed‐shell and radical molecular ions sheds light on their respective formation energetics, while variations in their respective infrared absorption bands evidence changes in intramolecular organization as the foldamer becomes more compact. These differences are compatible with the advent of radical‐pairing interactions.

Masstodon: A Tool for Assigning Peaks and Modeling Electron Transfer Reactions in Top-Down Mass Spectrometry.

Top-down mass spectrometry methods are becoming continuously more popular in the effort to describe the proteome. They rely on the fragmentation of intact protein ions inside the mass spectrometer. Among the existing fragmentation methods, electron transfer dissociation is known for its precision and wide coverage of different cleavage sites. However, several side reactions can occur under electron transfer dissociation (ETD) conditions, including nondissociative electron transfer and proton transfer reaction. Evaluating their extent can provide more insight into reaction kinetics as well as instrument operation. Furthermore, preferential formation of certain reaction products can reveal important structural information. To the best of our knowledge, there are currently no tools capable of tracing and analyzing the products of these reactions in a systematic way. In this Article, we present in detail masstodon: a computer program for assigning peaks and interpreting mass spectra. Besides being a general purpose tool, masstodon also offers the possibility to trace the products of reactions occurring under ETD conditions and provides insights into the parameters driving them. It is available free of charge under the GNU AGPL V3 public license.

Top-Down Characterization of Proteins with Intact Disulfide Bonds Using Activated-Ion Electron Transfer Dissociation.

Here we report the fragmentation of disulfide linked intact proteins using activated-ion electron transfer dissociation (AI-ETD) for top-down protein characterization. This fragmentation method is then compared to the alternative methods of beam-type collisional activation (HCD), electron transfer dissociation (ETD), and electron transfer and higher-energy collision dissociation (EThcD). We analyzed multiple precursor charge states of the protein standards bovine insulin, α-lactalbumin, lysozyme, β-lactoglobulin, and trypsin inhibitor. In all cases, we found that AI-ETD provides a boost in protein sequence coverage information and the generation of fragment ions from within regions enclosed by disulfide bonds. AI-ETD shows the largest improvement over the other techniques when analyzing highly disulfide linked and low charge density precursor ions. This substantial improvement is attributed to the concurrent irradiation of the gas phase ions while the electron-transfer reaction is taking place, mitigating nondissociative electron transfer, helping unfold the gas phase protein during the electron transfer event, and preventing disulfide bond reformation. We also show that AI-ETD is able to yield comparable sequence coverage information when disulfide bonds are left intact relative to proteins that have been reduced and alkylated. This work demonstrates that AI-ETD is an effective fragmentation method for the analysis of proteins with intact disulfide bonds, dramatically enhancing sequence ion generation and total sequence coverage compared to HCD and ETD.

Where Ion Mobility and Molecular Dynamics Meet To Unravel the (Un)Folding Mechanisms of an Oligorotaxane Molecular Switch.

At the interface between foldamers and mechanically interlocked molecules, oligorotaxanes exhibit a spring-like folded secondary structure with remarkable mechanical and physicochemical properties. Among these properties, the ability of oligorotaxanes to act as molecular switches through controlled modulations of their spatial extension over (un)folding dynamics is of particular interest. The present study aims to assess and further characterize this remarkable feature in the gas phase using mass spectrometry tools. In this context, we focused on the [4]5NPR+12 oligorotaxane molecule complexed with PF6- counterion and probed its co-conformational states as a function of the in-source-generated charge states. Data were interpreted in light of electronic secondary structure computations at the PM6 and DFT levels. Our results highlight two major co-conformational groups associated either with folded compact structures, notably stabilized by intramolecular π-π interactions and predominant for low charge states or with fully stretched structures resulting from significant Coulombic repulsions at high charge states. Between, the oligorotaxane adopts intermediate folded co-conformations, suggesting a stepwise unfolding pathway under increasing repulsive Coulombic constraints. The reversibility of this superstructural transition was next interrogated under electron-driven (nondissociative electron transfer) and heat-driven (collision-induced unfolding) activation stimuli. The outcomes support the feasibility to either unfold or (partially) refold the oligorotaxane foldamer on purpose in the gas phase. Our results show that the balance between the stabilizing π-π interactions and the versatile Coulomb interactions dictates the elongation state of the foldamer in the gas phase and emphasizes the adequacy of mass spectrometry tools for the superstructural characterization of desolvated prototypical artificial molecular machines.

Conformational Space and Stability of ETD Charge Reduction Products of Ubiquitin.

Owing to its versatility, electron transfer dissociation (ETD) has become one of the most commonly utilized fragmentation techniques in both native and non-native top-down mass spectrometry. However, several competing reactions-primarily different forms of charge reduction-occur under ETD conditions, as evidenced by the distorted isotope patterns usually observed. In this work, we analyze these isotope patterns to compare the stability of nondissociative electron transfer (ETnoD) products, specifically noncovalent c/z fragment complexes, across a range of ubiquitin conformational states. Using ion mobility, we find that more extended states are more prone to fragment release. We obtain evidence that for a given charge state, populations of ubiquitin ions formed either directly by electrospray ionization or through collapse of more extended states upon charge reduction, span a similar range of collision cross-sections. Products of gas-phase collapse are, however, less stabilized towards unfolding than the native conformation, indicating that the ions retain a memory of previous conformational states. Furthermore, this collapse of charge-reduced ions is promoted if the ions are 'preheated' using collisional activation, with possible implications for the kinetics of gas-phase compaction. Graphical Abstract ᅟ.

Understanding reaction pathways in top-down ETD by dissecting isotope distributions: A mammoth task

Electron transfer dissociation (ETD) is becoming increasingly more important in mass spectrometry-based structural proteomics due to its ability to cleave the protein backbone without annihilating the higher-order structure. Concomitant with ETD, non-dissociative charge reduction occurs primarily by proton transfer from the protein precursor to the ETD reagent, and by non-dissociative electron transfer from the reagent to the protein. While it has been known for some time that the preference for either proton or electron transfer is largely a function of the properties of the reagent, the extent of dissociative versus non-dissociative electron transfer is also dependent on instrument choice, precursor charge state and conformation, as well as ion activation before, during, and after cation/anion interaction. In this work, we use a novel software named MASSTODON to quantify the different reaction pathways in top-down spectra of ubiquitin, acquired on two different instruments: a Synapt G2 (Waters) and an LTQ Orbitrap Velos (Thermo). We compare the behavior of different precursor charge states, representing more folded or more extended conformations, and also evaluate the effect of increasing supplemental activation (on the Synapt) and reaction time (on the Orbitrap). In agreement with previous work, increasing supplemental activation promotes fragment release and hydrogen radical migration from (N-terminal) c fragments to (C-terminal) z fragments. A longer reaction time leads to increasingly more extensive charge reduction and a decrease in the relative occurrence of non-dissociative electron transfer compared to proton transfer, possibly indicating some degree of time- and charge-state dependent loss of electrostatic interactions on a timescale of a few tens of milliseconds.

Activated Ion Electron Transfer Dissociation for Improved Fragmentation of Intact Proteins.

Here we report the first implementation of activated ion electron transfer dissociation (AI-ETD) for top down protein characterization, showing that AI-ETD definitively extends the m/z range over which ETD can be effective for fragmentation of intact proteins. AI-ETD, which leverages infrared photon bombardment concurrent to the ETD reaction to mitigate nondissociative electron transfer, was performed using a novel multipurpose dissociation cell that can perform both beam-type collisional dissociation and ion-ion reactions on an ion trap-Orbitrap hybrid mass spectrometer. AI-ETD increased the number of c- and z-type product ions for all charge states over ETD alone, boosting product ion yield by nearly 4-fold for low charge density precursors. AI-ETD also outperformed HCD, generating more matching fragments for all proteins at all charge states investigated. In addition to generating more unique fragment ions, AI-ETD provided greater protein sequence coverage compared to both HCD and ETD. In all, the effectiveness of AI-ETD across the entirety of the m/z spectrum demonstrates its efficacy for robust fragmentation of intact proteins.

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS.

Non-dissociative charge reduction, typically considered to be an unwanted side reaction in electron transfer dissociation (ETD) experiments, can be enhanced significantly in order to reduce the charge state of intact protein complexes to as low as 1+ on a commercially available Q-IM-TOF instrument. This allows for the detection of large complexes beyond 100,000 m/z, while at the same time generating top-down ETD fragments, which provide sequence information from surface-exposed parts of the folded structure. Optimization of the supplemental activation has proven to be crucial in these experiments and the charge-reduced species are most likely the product of both proton transfer (PTR) and non-dissociative electron transfer (ETnoD) reactions that occur prior to the ion mobility cell. Applications of this approach range from deconvolution of complex spectra to the manipulation of charge states of gas-phase ions.

Electron transfer processes of atomic and molecular doubly charged ions: information from beam experiments

Single-electron transfer reactions in collisions of atomic and molecular doubly charged ions, with atoms and molecules, were investigated in a series of crossed-beam scattering, translational spectroscopy and product luminescence experiments. Investigation of a series of atomic dication-atom electron transfer at collision energies of 0.1–10 eV provided data on differential and relative total cross sections of state-to-state processes. Populations of electronic and vibrational states and rotational temperatures of molecular product ions were obtained from studies of non-dissociative electron transfer in systems containing simple molecular dications and/or molecular targets. The product electronic states populated with highest probability were those for which the translational energy release was 3–5 eV, indicating that the ‘reaction window' concept, based on the Landau–Zener formalism, is applicable also to molecular systems. Population of the vibrational states of the molecular products could be described by Franck–Condon factors of the vertical transitions between the reactant and product states, especially at higher (keV) collision energies. Rotational temperature of the product molecular cations was found to be surprisingly low, mostly 400–500 K, practically the temperature of the ion source.

A novel approach for quantitative peptides analysis by selected electron transfer reaction monitoring

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) with selective reaction monitoring (SRM) is a selective and sensitive method for quantitation of peptides. SRM is achieved via MS/MS utilizing collision-induced dissociation (CID) while monitoring unique precursor-product ion transitions. Low-energy CID tandem mass spectrometry has been, by far, the most common method used to dissociate peptide ions for sequence analysis. However, collisional scattering of product ions in CID results in decreased intensity of the primary product ion. The lower intensity of the targeted product ion can lead to a reduction in the sensitivity of a quantitative method that uses SRM. Electron transfer dissociation (ETD) is a fragmentation method that is complementary to CID. During the ETD reaction for doubly protonated peptides ([M+2H] 2+), there is a significant shift toward nondissociative electron transfer (ET) product species ([M+2H] + ). We utilized that particular defect in ETD to develop a new quantitative method for monitoring the transition of unique precursors ([M+2H] 2+) to charge-reduced ions ([M+2H] + ). We refer to this method as selective electron transfer reaction monitoring (SETRM). In ESI-MS, trypsin-digested peptides tend to generate doubly protonated peptide precursors. We found that SETRM was more suitable than SRM for these doubly charged tryptic peptides with nano-LC–MS/MS. The quantitative capabilities of SETRM provide a more sensitive way of performing quantitative experiments using the same instrument, thereby improving the application of electron transfer dissociation in proteomics.

Crossed-Beam Scattering Studies of Electron-Transfer Processes between the Dication CO22+ and Neutral CO2: Electronic States of Reactants and Products Involved

Crossed-beam scattering experiments were carried out at collision energies of 4.51 and 2.71 eV to elucidate the electronic states involved in the nondissociative and dissociative electron-transfer reactions observed following CO(2)(2+)/CO(2) collisions. Specifically, we focus on the observation that, in the dissociative electron-transfer reaction, forming CO(+), the majority of the CO(+) product ions are formed via electron capture by the CO(2)(2+) rather than via ejection of an electron from the neutral CO(2) reaction partner. The main channels resulting in nondissociative electron transfer are reactions of the ground (X(3)Sigma(g)(-)) and excited states of CO(2)(2+) to give different combinations of the ground and excited states of the product pair of CO(2)(+) ions in which the combination AA appears to be significant. The CO(+) ions appear mainly to arise from slow dissociation of CO(2)(+)(b(4)Pi(u)) formed following electron capture by the ground state of the dication reactant (X(3)Sigma(g)(-)), with possible contributions from electron capture by higher triplet excited states of the dication.

Performance Characteristics of Electron Transfer Dissociation Mass Spectrometry

We performed a large scale study of electron transfer dissociation (ETD) performance, as compared with ion trap collision-activated dissociation (CAD), for peptides ranging from approximately 1000 to 5000 Da (n approximately 4000). These data indicate relatively little overlap in peptide identifications between the two methods ( approximately 12%). ETD outperformed CAD for all charge states greater than 2; however, regardless of precursor charge a linear decrease in percent fragmentation, as a function of increasing precursor m/z, was observed with ETD fragmentation. We postulate that several precursor cation attributes, including peptide length, charge distribution, and total mass, could be relevant players. To examine these parameters unique ETD-identified peptides were sorted by length, and the ratio of amino acid residues per precursor charge (residues/charge) was calculated. We observed excellent correlation between the ratio of residues/charge and percent fragmentation. For peptides of a given residue/charge ratio, there is no correlation between peptide mass and percent fragmentation; instead we conclude that the ratio of residues/charge is the main factor in determining a successful ETD outcome. As charge density decreases so does the probability of non-covalent interactions that can bind a newly formed c/z-type ion pair. Recently we have described a supplemental activation approach (ETcaD) to convert these non-dissociative electron transfer product ions to useful c- and z-type ions. Automated implementation of such methods should remove this apparent precursor m/z ceiling. Finally, we evaluated the role of ion density (both anionic and cationic) and reaction duration for an ETD experiment. These data indicate that the best performance is achieved when the ion trap is filled to its space charge limit with anionic reagents. In this largest scale study of ETD to date, ETD continues to show great promise to propel the field of proteomics and, for small- to medium-sized peptides, is highly complementary to ion trap CAD.

Electron transfer and bond-forming reactions following collisions of Cl2+ and HCl2+ with CO

Collisions between Cl(2+) and CO have been investigated using time-of-flight mass spectrometry over a collision energy range between 2.2 eV and 7.1 eV in the centre-of-mass frame. The formation of Cl(+), CO(+) and C(+) in electron transfer reactions has been detected and an unusual bond-forming reaction which generates CCl(2+) has also been observed. The reactive cross-sections, in arbitrary units, for the electron transfer reactions have been evaluated. To extract these cross sections we employ a new method of analysing mass spectral intensities for crossed-beam experiments, an algorithm which allows inter-comparison of the fluxes of all the ionic products from the electron transfer reactions. The observed electron transfer reactivity has been rationalized by calculations based on Landau-Zener theory. To account for the observation of CCl(2+), we have calculated the relevant energetics showing that the lowest lying doublet state of this dication is bound and is energetically accessible at our collision energies. These energetic arguments indicate that electron transfer in the exit channel between the separating CCl(2+) and O atom probably forms C(+) ions via the dissociation of CCl(+). Additionally, collisions between HCl(2+) and CO have been studied at collision energies from 2.2 to 7.0 eV in the centre-of-mass frame. In this collision system, proton transfer to form HCO(+) is observed to compete efficiently with dissociative and non-dissociative electron transfer.

The formation of NO+ from the reaction of N22+ with O2

We have studied the potentially ionospherically significant reaction between N(2)2+ with O2 using position-sensitive coincidence spectroscopy. We observe both nondissociative and dissociative electron transfer reactions as well as two channels involving the formation of NO+. The NO+ product is formed together with either N+ and O in one bond-forming channel or O+ and N in the other bond-forming channel. Using the scattering diagrams derived from the coincidence data, it seems clear that both bond-forming reactions proceed via a collision complex [N2O2]2+. This collision complex then decays by loss of a neutral atom to form a daughter dication (NO2(2+) or N2O2+), which then decays by charge separation to yield the observed products.

The dynamics and kinematics of the electron transfer reactions of CF[image omitted] with Ar

The electron transfer reactions between CF [image omitted] and Ar have been studied using a position-sensitive coincidence time-of-flight mass spectrometer. The spectra show the formation of CF [image omitted], CF [image omitted] and CF [image omitted] ions, accompanied by Ar+ ions, by dissociative and non-dissociative electron transfer reactions. Analysis of the PSCO data shows that the CF [image omitted] and CF [image omitted] ions are formed by reactions of ground state CF [image omitted](2B2) ions. The CF [image omitted] product is formed in high lying vibrational states of its ground electronic state, just below the dissociation asymptote to [image omitted]. The formation of CF [image omitted] proceeds via a sequential mechanism, involving the population of highly excited vibrational levels of the ground electronic state of CF [image omitted] which dissociate to CF [image omitted](2A1) + F when the CF [image omitted] is well separated from the Ar+ ion. No evidence of complexation is observed. The energy dependence of the experimental yield of the CF+ ion is consistent with it arising from a reaction of an electronic excited state of CF [image omitted], as proposed in a previous study of this collision system. The unimolecular dissociation of the reactant dications is also observed and indicates that these ions are predominantly in their ground (2B2) electronic state. The kinetic energy release of this unimolecular reaction indicates the metastable well of the CF [image omitted](2B2) state is approximately 3 eV deep and also shows that CF [image omitted](2B2) ions well below the barrier to charge separation can dissociate on the time-scale of the experiment. This observation implicates predissociation, possibly via a repulsive dication state, as the mechanism for the unimolecular charge separation of the ground state of CF [image omitted].

Gas phase anion–molecule chemistry of SF6 , SeF6 and TeF6

The rate coefficients and anion product branching ratios for the thermal (300 K) reactions of O2− , O−, OH−, CF3− , and F− with SeF6 and TeF6 are reported. Non-dissociative and dissociative electron transfer pathways are identified, together with F+ abstraction and association. F− does not react with SeF6 ; all the other reactions proceed at or close to the capture rate. This is in contrast to SF6 , for which the only observed reaction with the above anions is slow (k = 3.0 × 10−11 cm3 molecule−1 s−1) electron transfer with O2− to form SF6− . The results are shown to be consistent with the available thermochemistry. There is evidence for a substantial intrinsic barrier to F+ abstraction, for this pathway is only observed when it is highly exothermic. There is a remarkably fast association reaction between F− and TeF6 , arising from the high fluoride affinity of TeF6 and facile expansion of the Te co-ordination sphere to accommodate seven ligands.

Insight into interfacial coordination chemistry through EPR spectroscopy

The application of EPR techniques to investigate the structure of surface complexes of selected transition metal ions and their interaction with small molecules are reviewed using the concepts of interfacial coordination chemistry. Such complexes act as catalytically active sites on surfaces and exhibit distinct features and reactivity. The bonding and activation pathways of small molecules include molecular adsorption, activation via non-dissociative and dissociative electron transfer as well as electroprotic chemical transformation. Complications arising from the low symmetry of surface complexes are also discussed.

Photosensitized Reduction of Sulfonium Salts: Evidence for Nondissociative Electron Transfer

The photosensitized reductions of 4-cyanobenzylmethylphenyl sulfonium tetrafluoroborate (1-BF4) by 9-phenylanthracene, 2-ethyl-9,10-dimethoxyanthracene, or perylene excited singlet states gives quantitative yields of phenyl methyl sulfide. Fluorescence quenching and the quantum yields for product formation, as functions of [1-BF4], give bimolecular rate constants (0.58−1.6 × 1010 M-1 s-1) for reaction of the excited sensitizers with the sulfonium salt. The limiting quantum yields, corresponding to infinite [1-BF4], are 0.65−0.77 for the three sensitizers, revealing significant inefficiencies in the photoreduction. These inefficiencies are assigned to the partitioning of a sulfuranyl radical intermediate in a two-step associative electron-transfer mechanism.

Isotope effects in the reaction of CF 22+ with H 2/D 2

The reaction between X 2 (XH, D) and CF 2 2+ has been investigated using a crossed beam spectrometer. The relative yields of the bond-forming reaction which produces XCF 2 + and the non-dissociative electron transfer reaction, which forms CF 2 +, have been determined as a function of the collision energy. These relative yields indicate the probability of forming XCF 2 + is independent of which isotope of hydrogen is the collision partner, a result consistent with a direct reaction mechanism.

Selected ion flow tube studies of the gas-phase reactions of O ·−, O 2·− and OH − with a variety of brominated compounds

The reactions of fourteen bromine-containing molecules, CH 3Br, CH 2Br 2, CHBrCl 2, CHBr 2Cl, C 2H 5Br, CH 2BrCH 2Br, CH 2BrCH 2Cl, CF 3Br, CF 2Br 2, CFBr 3, CBrClF 2, CFBr 2Cl, CCl 3Br and CF 2BrCF 2Br, with the anions O ·−, O 2 ·− and OH − in a 0.5 Torr helium buffer gas have been studied at 300 K using a selected ion flow tube. Reaction rate coefficients and branching ratios are presented. Several distinct reaction processes are observed among the large number of reactions studied, including nucleophilic attack, proton transfer, abstraction, and dissociative and non-dissociative electron transfer (within or out of a reaction complex). Of the forty-two bimolecular reactions, the rates of only two are significantly below the collisional rate and, for these two, competitive three-body processes are observed. Many of the reactions reported here have not previously been studied. This study, therefore, presents new data which provide additional insight into the reactions of anions with halogenated species.