Effects of ion/ion proton transfer reactions on conformation of gas-phase cytochrome <Emphasis Type="Italic">c</Emphasis> ions

Positive ions from cytochrome c are studied in a 3-D ion trap/ion mobility (IM)/quadrupole-time-of-flight (TOF) instrument with three independent ion sources. The IM separation allows measurement of the cross section of the ions. Ion/ion reactions in the 3-D ion trap that remove protons cause the cytochrome c ions to refold gently without other degradation of protein structure, i.e., fragmentation or loss of heme group or metal ion. The conformation(s) of the product ions generated by ion/ion reactions in a given charge state are similar regardless of whether the cytochrome c ions are originally in +8 or +9 charge states. In the lower charge states (+1 to +5) cytochrome c ions made by the ion/ion reaction yield a single IM peak with cross section of ～1110 to 1180 Ų, even if the original +8 ion started with multiple conformations. The conformation expands slightly when the charge state is reduced from +5 to +1. For product ions in the +6 to +8 charge states, ions created from higher charge states (+9 to +16) by ion/ion reaction produce more compact conformation(s) in somewhat higher abundances compared with those produced directly by the electrospray ionization (ESI) source. For ions in intermediate charge states that have a variety of resolvable conformers, the voltage used to inject the ions into the drift tube, and the voltage and duration of the pulse that extracts ions from the ion trap, can affect the observed abundances of various conformers.     

Study of metal ion labeling of the conformational and charge states of lysozyme by ion mobility mass spectrometry

The efficiency of Zn(2+), Cu(2+), Ni(2+), Co(2+), Fe(2+) or Mn(2+) labeling of the conformational and charge states of lysozyme was studied in H(2)O solvent at pH 2.5-6.8. Labeling of lysozyme was conducted with 50 M, 100 M and 500 M excess of the metal ion, resulting in the number of metal ions attached to lysozyme increasing two-fold over this range. At pH 6.2-6.8, Zn(2+), Cu(2+), Ni(2+), Co(2+) and Mn(2+) labeled the highly folded 7+ conformer and the 8+ and 9+ partially unfolded conformers of lysozyme with the same number of metal ion tags, with only Fe(2+) exhibiting no labeling. Lysozyme conserved its charge after metal ion labeling which shows at each charge state the divalent metal ion is replacing two protons. As the pH is lowered to 4.7-5.0 and 2.5-2.9, the labeling of lysozyme by Zn(2+), Cu(2+), Ni(2+), Co(2+) or Mn(2+) decreased in efficiency due to increased competition from protons for the aspartate and glutamate binding sites. The metal ions preferentially labeled the highly folded 7+ and partially unfolded 8+ conformers, but labeling decreased as the charge of lysozyme increased. In contrast to the other metal ions, Fe(2+) exhibited labeling of lysozyme only at the lowest pH of 2.8. At higher pH, the oxidation of Fe(2+) and formation of hydroxy-bridged complexes probably make the Fe(2+) unreactive towards lysozyme.     

A fast flow tube study of gas phase H/D exchange of multiply protonated ubiquitin

An electrospray ionization (ESI)/fast-flow technique has been applied to the study of gas phase hydrogen/deuterium (H/D) exchange kinetics. Multiply charged ubiquitin ions [ubiquitin + nH](n)(+), in charge states n = 7-13, were reacted with ND(3). The behavior of ND(3) as exchange reagent is different from that of the previously studied reagents, D(2)O and CH(3)OD. Contrary to those, the maximum number of exchanged hydrogen atoms and the overall exchange rate were observed to increase with increasing charge state of the ubiquitin ions. The results are reagent-dependent because the exchange mechanisms are different for the different reagents. This observation is in agreement with a recent conclusion by Beauchamp and co-workers that contrary to the assumption often expressed in earlier studies, H/D exchange kinetics may not directly reflect ion structures. The results for all three reagents are, however, consistent with observations of previous ion mobility experiments that with increasing charge state the conformers change from more compact, partially folded structures to elongated nearly linear ones. H/D exchange of (ubiquitin + 13H)(13+) with ND(3) leads to two separated ion populations reflecting the possible existence of two conformers with different exchange rates. The ions (ubiquitin + 8H)(8+) and (ubiquitin + 11H)(11+) represent a partially folded structure and an unfolded structure, respectively, and were studied in greater detail. The relative abundances of ions were measured in steps of 0.5 m/z (mass-to-charge ratio), as a function of the ND(3) flow rate. The experimental results were simulated by computer fitted curves based on a recently developed algorithm. The algorithm allows the extraction of sets of grouped rate constants. Eight rate constant groups were deduced for each of the two ions. These rate constants correspond to 32 and 44 H/D exchanges for the 8+ and 11+ charged ions, respectively. The results indicate higher individual rates for most of the exchanged atoms in the 11+ ion compared to the 8+ ion.     

Ion kinetic energy modulation for improved ion trapping in electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

A new technique for manipulating the kinetic energy distribution of electrospray ions that arrive at a Fourier transform ion cyclotron resonance trapped-ion cell is presented. Narrow kinetic energy distributions can complicate the selection of appropriate trapping conditions for electrospray ions and introduce charge discrimination in resulting mass spectra. Modulation of the applied skimmer potential controllably broadens the kinetic energy distribution, which improves the reproducibility of acquired spectra and eliminates charge discrimination. Mass spectra of horse heart cytochrome c are presented to demonstrate the utility of the technique. For example, applied static skimmer potentials of 12 and 9 V yield charge state distributions ranging from [M+19H]⁺¹⁹ to [M+12H]⁺¹² and [M+15H]⁺¹⁵ to [M+7H]⁺⁷, respectively. A 12 ± 2 V, 100-Hz modulation of the skimmer potential yields an electrospray spectrum with charge states that range from [M+19H]⁺¹⁹ to [M+7H]⁺⁷, which is more representative of the source distribution.     

Observation and implications of high mass-to-charge ratio ions from electrospray ionization mass spectrometry

High mass-to-charge ratio ions (> 4000) from electrospray ionization (ESI) have been observed for several proteins, including bovine cytochrome c (M _r 12,231) and porcine pepsin (M _r 34,584), by using a quadrupole mass spectrometer with an m/z 45,000 range. The ESI mass spectrum for cytochrome c in an aqueous solution gives a charge state distribution that ranges from 12 + to 2 +, with a broad, low-intensity peak in the mass-to-charge ratio region corresponding to the [M + H]⁺ ion. the negative ion ESI mass spectrum for pepsin in 1% acetic acid solution shows a charge state distribution ranging from 7? to 2?. To observe the [M - H]^? ion, harsher desolvation and interface conditions were required. Also observed was the abundant aggregation of the protens with average charge states substantially lower than observed for their monomeric counterparts. The negative ion ESI mass spectrum for cytochrome c in 1–100 mM NH₄OAc solutions showed greater relative abundances for the higher mass-to-charge ratio ions than in acuidic solutions, with an [M - H]^? ion relative abundance approximately 50% that of the most abundant charge state peak. The observation that protein aggregates are formed with charge states comparable to monomeric species (at fower mass-to-charge ratios) suggests that the high mass-to-charge ratio monomers may be formed by the dissociation of aggregate species. The observation of low charge state and aggregate molecular ions concurrently with highly charged species may serve to support a variation of the charged residue model, originally described by Dole and co-workers (Dole, M., et al. J. Chem. Phys. 1968, 49, 2240; Mack, L. L., et al. J. Chem. Phys. 1970, 52, 4977) which involves the Coulombically driven formation of either very highly solvated molecular ions or lower ananometer-diameter droplets.     

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin +7 ions are converted to lower charge states down to +1; the ions in lower charge states tend to be in compact conformations with cross sections down to ～880 Ų. The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin +7 and +6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described.     

Targeted ion parking for the quantitation of biotherapeutic proteins: Concepts and preliminary data

Targeted ion parking (or TIPing) is the first quantitative application of ion/ion reactions for mass spectrometry. In TIPing, intact biotherapeutic proteins are electrosprayed as intact molecules (no digestion) and, as expected, many multiply protonated species are produced (e.g., (M + 7H)⁷⁺, (M + 8H)⁸⁺, etc.). Several of these multiply charged species are selectively isolated using a quadrupole mass analyzer and then contained in a linear ion trap. The protein ions are then subjected to a proton-transfer reaction with a reagent anion. The ions undergo sequential charge reduction (e.g., to (M + 6H)⁶⁺) during a defined reaction period. Applying a low-amplitude waveform to the trap during this reaction time stops the ion/ion reaction at a chosen (and predicted) charge state for the protein. This funnels the analyte ions into a single channel with relatively high efficiency (>-50% of reactant ion signal is converted into product ion signal) that can be used for quantitation. In TIPing, the target protein’s molecular weight and charge state distribution are the only prerequisite knowledge required. This information can be acquired experimentally or can be easily predicted based upon amino acid sequences. Preliminary data for a biotherapeutic protein, a domain antibody, were collected using TIPing coupled online with liquid chromatography (LC-TIPing). The LC-TIPing data demonstrate a linear response for samples from 10–1000 ng/mL extracted from a complex plasma sample, demonstrating the analytical potential for TIPing.     

High-frequency fourier transform ion cyclotron resonance mass spectrometry

The experimental Fourier transform ion cyclotron resonance (FT/ICR) frequency range has been extended to 107 MHz. We report the observation of FT/ICR signals from electron-ionized species of mass-to-charge ratio 8, 7, 6, 5, 4, 3, 2, and 1 μ per elementary charge. We show that moderately high charge states of atomic ions (e.g., N³⁺) are easily generated and detected. Several applications for high-frequency FT/ICR mass spectrometry are proposed and discussed.     

Substituent effects on the gas-phase fragmentation reactions of sulfonium ion containing peptides

The multistage mass spectrometric (MS/MS and MS3) gas-phase fragmentation reactions of methionine side-chain sulfonium ion containing peptides formed by reaction with a series of para-substituted phenacyl bromide (XBr where X=CH2COC6H4R, and R=--COOH, --COOCH3, --H, --CH3 and --CH2CH3) alkylating reagents have been examined in a linear quadrupole ion trap mass spectrometer. MS/MS of the singly (M+) and multiply ([M++nH](n+1)+) charged precursor ions results in exclusive dissociation at the fixed charge containing side chain, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility). However, loss of the methylphenacyl sulfide side-chain fragment as a neutral versus charged (protonated) species was observed to be highly dependent on the proton mobility of the precursor ion, and the identity of the phenacyl group para-substituent. Molecular orbital calculations were performed at the B3LYP/6-31+G** level of theory to calculate the theoretical proton affinities of the neutral side-chain fragments. The log of the ratio of neutral versus protonated side-chain fragment losses from the derivatized side chain were found to exhibit a linear dependence on the proton affinity of the side-chain fragmentation product, as well as the proton affinities of the peptide product ions. Finally, MS3 dissociation of the nominally identical neutral and protonated loss product ions formed by MS/MS of the [M++H]2+ and [M++2H]3+ precursor ions, respectively, from the peptide GAILM(X)GAILK revealed significant differences in the abundances of the resultant product ions. These results suggest that the protonated peptide product ions formed by gas-phase fragmentation of sulfonium ion containing precursors in an ion trap mass spectrometer do not necessarily undergo intramolecular proton 'scrambling' prior to their further dissociation, in contrast to that previously demonstrated for peptide ions introduced by external ionization sources.     

Comparison of infrared multiphoton dissociation and collision-induced dissociation of supercharged peptides in ion traps

The number and types of diagnostic ions obtained by infrared multiphoton dissociation (IRMPD) and collision-induced dissociation (CID) were evaluated for supercharged peptide ions created by electrospray ionization of solutions spiked with m-nitrobenzyl alcohol. IRMPD of supercharged peptide ions increased the sequence coverage compared with that obtained by CID for all charge states investigated. The number of diagnostic ions increased with the charge state for IRMPD; however, this trend was not consistent for CID because the supercharged ions did not always yield the greatest number of diagnostic ions. Significantly different fragmentation pathways were observed for the different charge states upon CID or IRMPD with the latter yielding far more immonium ions and often fewer uninformative ammonia, water, and phosphoric acid neutral losses. Pulsed-Q dissociation resulted in an increase in the number of internal product ions, a decrease in sequence-informative ions, and reduced overall ion abundances. The enhanced sequence coverage afforded by IRMPD of supercharged ions was demonstrated for a variety of model peptides, as well as for a tryptic digest of cytochrome c.     

Ion-Ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.     

Single electron traps at the surface of polycrystalline MgO: assignment of the main trapping sites

Paramagnetic centers at the surface of ionic oxides in the form of trapped electrons can be generated by exposure of the material to alkali metal or hydrogen atoms or of molecular hydrogen under UV irradiation. For many years, it has been assumed that the resulting paramagnetic centers consist of oxygen vacancies filled by one electron. High-resolution electron spin resonance spectra and ab initio quantum chemical calculations show that the paramagnetic centers consist of (H(+))(e(-)) electron pairs formed at morphological irregularities of the surface. At least three different kinds of (H(+))(e(-)) centers, [A], [B], and [C], have been identified with abundances of 80%, 10%, and 8%, respectively. In this work, we compare a wide set of measured and computed g-factors and hyperfine coupling constants of the unpaired electron with the surrounding (25)Mg, (17)O, and (1)H nuclei and we propose a general assignment of the centers. (H(+))(e(-)) pairs formed at Mg(4c) ions at steps and edges account for species [A], centers formed at Mg(4c) ions at reverse corners correspond to species [B], and species [C] originates from (H(+))(e(-)) pairs formed at Mg(3c) ions at corners and kinks.     

Ion Trap Collision-Induced Dissociation of Human Hemoglobin α-Chain Cations

Multiply protonated human hemoglobin alpha-chain species, ranging from [M + 4H]4+ to [M + 20H]20+, have been subjected to ion trap collisional activation. Cleavages at 88 of the 140 peptide bonds were indicated, summed over all charge states, although most product ion signals were concentrated in a significantly smaller number of channels. Consistent with previous whole protein ion dissociation studies conducted under similar conditions, the structural information inherent to a given precursor ion was highly sensitive to charge state. A strongly dominant cleavage at D75/M76, also noted previously in beam-type collisional activation studies, was observed for the [M + 8H]8+ to [M + 11H]11+ precursor ions. At lower charge states, C-terminal aspartic acid cleavages were also prominent but the most abundant products did not arise from the D75/M76 channel. The [M + 12H]12+-[M + 16H]16+ precursor ions generally yielded the greatest variety of amide bond cleavages. With the exception of the [M + 4H]4+ ion, all charge states showed cleavage at the L113/P114 bond. This cleavage proved to be the most prominent dissociation for charge states [M + 14H]14+ and higher. The diversity of dissociation channels observed within the charge state range studied potentially provides the opportunity to localize residues associated with variants via a top-down tandem mass spectrometry approach.     

Peak deconvolution in high-field asymmetric waveform ion mobility spectrometry (FAIMS) to characterize macromolecular conformations

Protonated poly(ethylene glycol), produced by electrospray ionization (ESI), with molecular weights ranging from 0.3 to 5 kDa and charge states from 1+ to 7+ were characterized using high-field asymmetric waveform ion mobility spectrometry (FAIMS). Results for all but some of the 3+ and 4+ charge states are consistent with a single gas-phase conformer or family of unresolved conformers for each of these charge states. The FAIMS compensation voltage scans resulted in peaks that could be accurately fit with a single Gaussian for each peak. The peak widths increase linearly with compensation voltage for maximum ion transmission but do not depend on m/z or molecular weight. Fitting parameters obtained from the poly(ethylene glycol) data were used to analyze conformations of oxidized and reduced lysozyme formed from different solutions. For oxidized lysozyme formed from a buffered aqueous solution, a single conformer (or group of unresolved conformers) was observed for the 7+ and 8+ charge states. Two conformers were observed for the 9+ and 10+ charge states formed from more denaturing solutions. Data for the fully reduced form indicate the existence of up to three different conformers for each charge state produced directly by ESI and a general progression from a more extended to a more folded structure with decreasing charge state. These results are consistent with those obtained previously by proton-transfer reactivity and drift tube ion mobility experiments, although more conformers were identified for the fully reduced form of lysozyme using FAIMS.     

Modeling of the gas-phase ion chemistry of protonated arginine

Arginine is often involved at the C-terminus of peptides obtained from tryptic digests of proteins. The very basic guanidine group of the side-chain of arginine has a large effect on the backbone fragmentation of protonated peptides. Furthermore, arginine exhibits specific fragmentation reactions involving its side-chain. Various tautomerization states, conformers and side-chain dissociation channels of protonated arginine were studied using theoretical methods. The guanidine loss of protonated arginine is proved to be an S(N)2 substitution on the delta-carbon of the side-chain, starting from species containing the N(epsilon)H-C(+)(N(eta)H(2))(N(eta')H(2)) or -N(epsilon) (+)H(2)-C(N(eta)H)(N(eta')H(2)) moieties and leads to formation to either protonated guanidine or protonated proline. In the corresponding transition structures the proline moiety is protonated. Under low-energy collision conditions the extra proton transfers to the guanidine moiety, leading to the formation of C(+)(NH(2))(3). On the other hand, the lifetime of the fragmenting species under high-energy collision conditions is shorter, resulting in enhanced formation of protonated proline and its dissociation products. The first step of ammonia loss is the leaving of a preformed NH(3) from tautomers containing the -N(epsilon)H-C(N(eta)H(3) (+))(N(eta')H) or -N(epsilon)-C(N(eta)H(3) (+))(N(eta')H(2)) moieties. The resulting protonated carbodiimide group can be stabilized by intramolecular nucleophilic attack, leading to ring formation. Overall, reactions involved in the ammonia loss from protonated arginine can be considered as an S(N)1 substitution on the central zeta-carbon of the guanidine group.     

氯代甲苯双电荷离子的单分子解离反应研究

研究了在70 eV电子轰击电离条件下，氯代甲苯及氯化苄产生的双电荷离子[C_7H_7Cl]~(2＋)、[C_7H_6Cl]~(2＋·)和[C_7H_5Cl]~(2＋)为母体的两种类型单分子解离反应．主要讨论了亚稳双电荷离子的异构化反应、失H解高的“偶电子规则”以及单分子电荷分离过渡态的结构．     

Ultrafast excited state dynamics controlling photochemical isomerization of N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium coordinated to a {Re I(CO)3(2,2'-bipyridine)} chromophore

Two multifunctional photoactive complexes [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) and [Re(MeDpe(+))(CO)(3)(bpy)](2+) (MeDpe(+)=N-methyl-4-[trans-2-(4-pyridyl)ethenyl]pyridinium, bpy=2,2'-bipyridine) were synthesized, characterized, and their redox and photonic properties were investigated by cyclic voltammetry; ultraviolet-visible-infrared (UV/Vis/IR) spectroelectrochemistry, stationary UV/Vis and resonance Raman spectroscopy; photolysis; picosecond time-resolved absorption spectroscopy in the visible and infrared regions; and time-resolved resonance Raman spectroscopy. The first reduction step of either complex occurs at about -1.1 V versus Fc/Fc(+) and is localized at MeDpe(+). Reduction alone does not induce a trans-->cis isomerization of MeDpe(+). [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) is photostable, while [Re(MeDpe(+))(CO)(3)(bpy)](2+) and free MeDpe(+) isomerize under near-UV irradiation. The lowest excited state of [Re(Cl)(CO)(3)(MeDpe(+))(2)](2+) has been identified as the Re(Cl)(CO)(3)-->MeDpe(+ 3)MLCT (MLCT=metal-to-ligand charge transfer), decaying directly to the ground state with lifetimes of approximately 42 (73 %) and approximately 430 ps (27 %). Optical excitation of [Re(MeDpe(+))(CO)(3)(bpy)](2+) leads to population of Re(CO)(3)-->MeDpe(+) and Re(CO)(3)-->bpy (3)MLCT states, from which a MeDpe(+) localized intraligand (3)pipi* excited state ((3)IL) is populated with lifetimes of approximately 0.6 and approximately 10 ps, respectively. The (3)IL state undergoes a approximately 21 ps internal rotation, which eventually produces the cis isomer on a much longer timescale. The different excited-state behavior of the two complexes and the absence of thermodynamically favorable interligand electron transfer in excited [Re(MeDpe(+))(CO)(3)(bpy)](2+) reflect the fine energetic balance between excited states of different orbital origin, which can be tuned by subtle structural variations. The complex [Re(MeDpe(+))(CO)(3)(bpy)](2+) emerges as a prototypical, multifunctional species with complementary redox and photonic behavior.     

Charge state dependent collision-induced dissociation of native and reduced porcine elastase

The [M + 20H](20+)-[M + 12H](12+) charge states of native and reduced porcine elastase, a 25.9 kDa serine protease, were subjected to collisional activation in a quadrupole ion trap. For most charge states, ion parking was used to increase the number of parent ions over that yielded directly by electrospray. Ion-ion proton transfer reactions were used to reduce product ion charge states largely to +1 to simplify spectral interpretation. Both forms of the protein show charge state dependent fragmentation behavior. The native protein, which contains four disulfide linkages, shows almost no evidence for fragmentation within the regions of the protein linked by disulfide bonds. However, at the lowest charge states studied, evidence for cleavage of a least one of the disulfide bonds was evident in the appearance of a c-type ion. The highest charge states of native elastase showed several prominent cleavages C-terminal to valine residues. As the charge state decreased, however, preferential cleavages at acidic amino acid residues became important. The reduced form of the protein did not show particularly prominent cleavages at valine residues. However, many of the same preferential cleavages at acidic amino acid residues noted for the native protein were also observed in the same charge states of the reduced protein. The reduced protein also showed additional cleavages from regions of the protein that are ordinarily protected by disulfide linkages in the native form.     

Complexes of cadmium ion with guanine bases detected by electrospray ionization mass spectrometry

The complexations of cadmium ion with guanine bases were detected by electrospray ionization mass spectrometry (ESI-MS). In order to explore the toxicity of cadmium, such as oxidative stress to DNA and carcinogenesis, it is very important to determine the interactions between the cadmium ion and nucleotide. The analysis of mixed cadmium ion-guanosine aqueous solution (molar ratio 1 : 9) using ESI-MS (cone voltage 20 V) showed the presence of various cadmium complex ions, such as [n (guanosine) + Cd](2+) (n = 3-8), [2guanine + Cd](2+), [guanosine + guanine + Cd](2+) and [guanosine + 2guanine + Cd](2+). The observed [2guanine + Cd](2+), [guanosine + guanine + Cd](2+) and [guanosine + guanine + Cd](2+) ions are formed through the dissociation of the N-glycoside bond at the interface of ESI-MS. For deoxyguanosine and ethylguanine, similar cadmium complexes were observed. However, the complexes between the cadmium ion and 8-hydroxydeoxyguanosine were not detected. Furthermore, when a higher molar ratio (Cd : guanosine) or cone voltage were used, more of the monovalent ion peaks, such as [Cd(guanine - H)(2) + H](+) and [Cd(guanosine - H)(2) + H](+), were observed and a decrease in the abundance of the divalent ions, such as [n(guanosine)+Cd](2+), occurred.