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Neutral-particle emission from multiply charged biomolecular ions in collisions with electrons

Electrostatic storage rings are suitable for storing macromolecules, such as biomolecules. The combination of electrostatic storage rings and electron targets opens up a new aspect for studying electron-biomolecular ion collisions with well-defined energies in the gas-phase. Neutral-particle emissions in collisions of electrons with biomolecular ions, studied by the electrostatic storage rings with the electron targets, are reviewed. In electronbiomolecular anion collisions, high threshold energies for producing neutrals are a common remarkable feature. Meanwhile, in electron-biomolecular cation collisions, resonant neutral particle emissions were observed in some specific ions and at particular energies. The origins of these phenomena are discussed based on well-known concepts in physics, chemistry and biology.


Introduction
Since the late 1980s, electron-light molecular ion collisions have been studied extensively by using magnetic storage rings with electron cooling devices. The features of this method are summarized: high luminosity, high resolution, variable relative energies, low-background and quenching of vibrationally excited molecular ions. It is very attractive to expand the research object from light molecules to biomolecules. For this purpose, electrostatic storage rings (ESR) are more suitable than the conventional magnetic storage rings, because the electrostatic rigidity is independent of the ion mass, unlike the magnetic rigidity, and thus all ions with different masses are stored in the ring under the same operational conditions. An ESR was first constructed in Brookhaven National Laboratory for studying accelerator technology in the late 1950s [1], while an ESR aimed at the atomic physics was first built at the University of Aarhus [2] and then at the High Energy Accelerator Research Organization (KEK) [3], which was equipped with electron targets. Thus, these allow studies on electron-biomolecular ion collisions at well-defined energies. Another feature of the storage-ring technique is that neutral particles emitted in electron-ion collisions can be detected, which are not easily accessible with any other experimental setups.
Electron-DNA collisions are attracting much interest related to our understanding of elementary processes, radiation damage and radiation therapy. Collisions with electrons can cause severe damage to DNA molecules through direct and indirect strand breaks, and are crucial to the loss of genetic information. There have been many experiments on electron-neutral DNA collisions [4] and the collision-induced dissociation (CID) [5] of DNA anions in gas. However, little has been reported on electron-DNA ion collisions, probably due to a lack of experimental means. In this paper, electronoligonucleotide anion/cation collisions with the ESRs are reviewed.
Molecular dissociations in collisions with electrons are also important related to the structure analysis of peptides, proteins and their modified products. Specific bond cleavage in collisions allows the identification of the composition and sequence of structural elements in these molecules. In the atomic physics research field, the dissociative recombination (DR) for light molecular cations at low energies has been extensively studied for a long time. Besides, DR at high energies was found in 1993 [6,7,8]. Meanwhile, in the research field of the mass spectrometry, the electron capture dissociation (ECD) process [9] was found in 1998, which allows the adiabatic, non-ergodic fragmentation of peptides. Besides, the hot electron capture dissociation (HECD) process, which is ECD at high electron energies, was reported [10]. Both DR and ECD are very similar concepts, although they have been developed in different research fields. In this paper, the neutral resonances produced in electronprotonated peptide monocation collisions are compared with the resonant bond-cleavages in electronhighly protonated peptide cation collisions.

Experimental setup
In this section, the experimental methods at two institutes, University of Aarhus and KEK, are reviewed along with a possible neutral-particle detector. Figure 1 shows the experimental setup of the storage ring in Aarhus, called ELISA. Ions are produced by a cold-cathode ion source or an electrospray ion source (ESI), and injected into the ring after being momentum analyzed by a dipole magnet [12,13]. In order to amplify the intensity of ions from the ESI, an octupole ion trap was used. An electron target, called ETRAP, is in a crossing beam geometry, and its electron-beam diameter is about 1.5 cm. ETRAP is very compact and has a localized collision region between ion and electron beams. The electron energies were calibrated by comparing the threshold energies of neutral particle emissions in electron-anion collisions obtained by ELISA and a magnetic storage ring, ASTRID.  Figure 2 shows the experimental setup at KEK. Light ions are produced from an electron cyclotron resonance ion source, while biomolecular ions are produced in an ESI [14]. The ions from the ESI are first stored in octupole ion trap, ejected as a bunch and accelerated to 20 keV. Ions are then injected into the ESR after being mass-analyzed by an analyzer consisting of a spherical deflector and a dipole magnet. The electron target is in a merging beam geometry, and its structure is the same as the electron cooler with an expanded electron beam [15]. The length of the merging region between ions and electrons is 20 cm. A thick electron target allows operation with a low electron beam current on the order of 10 A, resulting in negligible space-charge depletion. The electron energies were calibrated by using the DR processes for light molecular ions, such as H 2 + , HD + and D 2 + .  Figure 2. Electrostatic storage ring equipped with an electrospray ion source, a mass spectrometer and an electron beam device at KEK.

Neutral-particle detectors
Neutral particles are detected by a micro-channel plate (MCP) installed in the vacuum extension for both storage rings. MCP is not sensitive to particle energies; moreover, its detection efficiencies decrease with a decrease in the ion velocity [16]; in other words, the increasing ion mass at a fixed incident ion energies. On the other hand, a new-type detector, which is a superconducting detector, was proposed [17]. It can detect neutral particles as well as ions and x-rays. Besides, the detector is sensitive to energies, thus resulting in possible mass measurements, since the velocities of neutral fragments are the same. It was also observed that the superconducting detector can measure particles having a mass of up to 1 MDa with an efficiency of 100% [18]. The detector is still in the development stage, but is promising for the future.

Electron-light anion collisions
In ELISA, electron-anion collisions have been extensively studied. Figure 3 shows the cross section for the formation of neutrals as a function of the electron kinetic energy for NO 2 -, and singly-and doubly-hydrated NO 2 - [19]. The cross sections clearly display dianion resonances, which shift to lower energies with the addition of water molecules. Thus, the addition of single water molecule to NO 2 increases the electron binding energy by 0.8 eV per water for the ground state dianion; namely, the dianion becomes less electronically unbound upon hydration. Furthermore, experiments were performed on dianion beams of Pt(CN) 4 2and Pt(CN) 6 2- [11]. These ions are essentially stable and have provided high thresholds of 17.2 and 18.7 eV for neutral production in the interaction between free electrons and these dianions. The relatively high thresholds reflect the strong Coulomb repulsion in the incoming channel. A trianion resonance was identified with a positive energy of 17.0 eV for the Pt(CN) 4 2square-planar complex, while three trianion resonances were identified for the Pt(CN) 6 2octahedral complex with positive energies of 15.3, 18.1 and 20.1 eV. In these analyses, the form of the non-resonant detachment cross section was modeled by a simple reaction-zone model, applicable for atomic anions. A detachment reaction takes place at a constant probability, p, if the incoming electron comes within a distance R from the anion. The non-resonant cross section is given by where a 0 is the Bohr radius and R is given by R 1/ E th . The generalized threshold energy, E th , is given by [11,20] E th 1.23 where E det is the electron detachment energy and Q is the anion charge.

Electron-biomolecular anion collisions
Mononucleotides are the building blocks of DNA and RNA, consisting of nucleotide bases, a sugar and a phosphate group. Collisions between electrons and deprotonated mononucleotides, AMPand UMP -, were studied in Aarhus [21]; the results are shown in figure 5. It is found that the threshold energy for electron-induced reactions leading to production of neutral fragments is as high as about 12 eV for both species. The high threshold is explained within the context of a classical reaction zone model and a high electron-binding energy of about 5 eV for the two mononucleotides. There is no evidence in the cross sections for the formation of short-lived doubly charged anions.
At KEK, neutral productions in the collisions of electrons with DNA oligomer anions were studied for oligonucleotide groups with the same charge, but different lengths and sequences [22]. The results are shown in figure 6. As can be seen, the threshold energies are almost independent of the length and sequence of the nucleotides, but depended strongly on the anion charge. The threshold energies appear to increase in steps of around 10 eV. If we try to explain this behavior by a simple Coulomb barrier, given by E CB~Q / r 0 , we have to assume an extremely small radius of 2.2 a.u., which is not probable because charges are distributed over a wide range of anions. Furthermore, the classical reaction zone model is not suitable to explain this phenomenon, because the threshold energies given by equation (2) are proportional to Q , while the experimental threshold energies are proportional to Q. On the other hand, earlier, it was speculated [23] that, when DNA is subject to direct hits by high-energy particles, collective electron excitations in the form of bulk plasmon could act as the first stage of DNA response, triggering successive events, and leading at last to DNA strand breaks. To support this idea, some studies appeared [24,25,26], which adopted the conventional solid-state viewpoint, and took into account only all of the valence electrons of the DNA bases. However, in this experimental setup, highenergy electrons hit separate DNA oligomer anions. Hence, it was instead assumed that all of the valence electrons of a separate finite oligonucleotide anion are involved in the response to electron hits by exhibiting plasmonlike oscillations. A plasmon is a quantum of collective oscillation of electrons in a metal, dielectrics, atom and molecule. The energy is given by n p . One quantum of plasmon energy is given by where n V is the density of the valence electrons, which is given by the total number of valence electrons divided by the DNA volume. The calculated plasmon energies are approximately on the order of 10 eV. In figure 6 the plasmon energies are compared with the experimental threshold energies. Interestingly, the experimental threshold energies approximately agree with the corresponding plasmon energies multiplied by an integer number (shown as triangles in figure 6), which is equal to the value of the corresponding DNA ion charge. In other words, higher electron hits seem to simultaneously excite a larger number of plasmon quanta. This phenomenon is not limited to DNA, but it can also be observed for peptide anions [27]. The threshold energies seem to be independent of the electron affinities.   Figure 7 shows the neutral particle production rate as a function of the relative energy in collisions of electrons with angiotensin I, II and III [28]. As can be seen in the figure, a slight, but clear, increase in the rates can be recognized at energies of less than 2 eV, which comes from DR at low energies. On the other hand, there are large bumps at around 6.5 eV with shoulders at around 9 eV. These peak energies are almost the same for each angiotensin ion. Besides, the rates greatly decrease with a decrease in the number of amino acid residues, despite a slight change. To explain the origin of these resonances, the same experiments were performed on some of the singly protonated amino-acids, which are constituents of angeotensins. However, no bump at all was found for these ions. That means the bumps do not come from amino-acids, but from peptide bonds. These are not special examples, but the resonance was also observed in other peptides at the same energies [29]. These resonances remind us of our earlier data on DR for HeH + [6], as shown in figure 8. In electron and light molecular-ion collisions, the DR at high energies was found for HeH + , HD + [7], H 3 + [8] and also for many heavier molecular ions, in addition to the DR at zero relative energy. These high-energy resonances were explained as dissociative recombination through two-electron excited states that form neutral Rydberg manifolds converging to the first and more highly excited sates of ions. Thus, the peak at 6.5 eV in figure 7 seems to come from a Feshbach resonance in which an incident electron promotes core electron excitations in the target cations, by losing their kinetic energy, and thus becoming captured. These highly excited resonance states should then have enough energy for molecular dissociation. If there is a high positive charge-density region in a peptide bond at a protonated site, a free electron excites a bonding electron from a state to a * state, and is captured in an autoionizing state, resulting in a two-electron excited state that can dissociate or re-ionize. The inelastic ion excitation process should start from a threshold of around 6.5 eV, corresponding to the * transition. The experimental rate, however, has a maximum in this energy region. This means that the peak at 6.5 eV comes from the DR process, while the shoulder at around 9 eV probably comes from a dissociative excitation process.  Meanwhile, in the research field of mass spectrometry, molecular dissociations in the collisions of electrons with peptide cations have been extensively studied [30,31]. Figure 9 shows the abundance vs electron energy in collisions of electrons with multiply protonated peptides measured by a Fourier transform (FT) mass spectrometer [10]. There are two big peaks in the high-energy region in addition to the peak at zero energy. These correspond to HECD and ECD, respectively. The low-and highenergy peaks for the HECD come from c-z and b-y cleavage of the peptide bonds, respectively. Interestingly, these peak energies almost agree with the results in figure 7. fragments; 1+: ( ) C-N bond cleavages [10].

Electron-oligonucleotide cation collisions
Electron-monocation collisions were also studied for DNA oligomers [27]. The mechanism and the extent of charging in ESI have been hotly debated. Furthermore, the charge position and distribution in ions produced in ESI are not quite clear. Under these circumstances, there is no other way of reasoning, except for assuming that the charge distributions in ions are dominated by the pH value of the solution. In the liquid phase with a pH of around 3.5, phosphates are deprotonated and negatively charged, while bases are protonated and positively charged. As a result of this charging, mono-cations seem to be produced. Interestingly, a large peak was found at around 4.5 eV as well as an increase in the rate toward zero relative energies for a trimer monocation with a sequence of d(AAA). This resonant neutral production seems to come from DR through a Feshbach resonance. Furthermore, it was found that the strengths of the resonances are strongly correlated with the base composition and sequence of nucleotide cations. In the DNA double helix, the two strands are known to be held together by hydrogen bonds between complementary bases. Meanwhile, there is also a non-negligible coupling between the nearest-neighbor bases along the same strand, which is referred to as base stacking. Both are important for stabilizing the DNA structure [32]. Strikingly, the observed tendencies in the strengths of the 4.5 eV-resonance bumps turn out to be correlated with the wellknown qualitative base-stacking trends, even in spite of possible perturbations in the stacking modalities due to base protonation. It is thus tempting to conclude that the neutral resonances can be associated with the presence of intramolecular base stacking in nucleotides [33].

Summary
A new way of studying electron-biomolecular ion collisions in the gas phase was opened by adding electron targets to electrostatic storage rings. Interesting phenomena have been found on neutralparticle emission from multiply charged biomolecular ions in collisions with electrons. Apparently, further experimental and theoretical studies are required to understand these phenomena more clearly. Nevertheless, these challenging experiments on biomolecules will surely provide a clue to unravel the problems lying between biological phenomena and fundamental physics.