Cross‐Talk Between the Octarepeat Domain and the Fifth Binding Site of Prion Protein Driven by the Interaction of Copper(II) with the N‐terminus

Prion diseases are a group of neurodegenerative diseases based on the conformational conversion of the normal form of the prion protein (PrP^C) to the disease‐related scrapie isoform (PrP^Sc). Copper(II) coordination to PrP^C has attracted considerable interest for almost 20 years, mainly due to the possibility that such an interaction would be an important event for the physiological function of PrP^C. In this work, we report the copper(II) coordination features of the peptide fragment Ac(PEG₁₁)₃PrP(60‐114) [Ac=acetyl] as a model for the whole N‐terminus of the PrP^C metal‐binding domain. We studied the complexation properties of the peptide by means of potentiometric, UV/Vis, circular dichroism and electrospray ionisation mass spectrometry techniques. The results revealed that the preferred histidyl binding sites largely depend on the pH and copper(II)/peptide ratio. Formation of macrochelate species occurs up to a 2:1 metal/peptide ratio in the physiological pH range and simultaneously involves the histidyl residues present both inside and outside the octarepeat domain. However, at increased copper(II)/peptide ratios amide‐bound species form, especially within the octarepeat domain. On the contrary, at basic pH the amide‐bound species predominate at any copper/peptide ratio and are formed preferably with the binding sites of His96 and His111, which is similar to the metal‐binding‐affinity order observed in our previous studies.     

Charge transfer study through the determination of the ionization energies of tetrapeptides X3-Tyr, X = Gly, Ala, or Leu. Influence of the inclusion of one glycine in alanine and leucine containing peptides

The energies of the fundamental and several excited states of tetrapeptide radical cations were determined at the outer valence Green's function (OVGF) level, at three geometries corresponding to the lowest energy conformations: two for the neutral and one for the cation. The conformations were optimized at the density functional theory level within the B3LYP framework. It was found that, from a purely energetic point of view, a charge initially created on the tyrosine chromophore could migrate without any geometrical change and without further activation once the excited electronic state of the ionized chromophore was formed. This migration could reach the NH(2) terminus for the neutral conformations but should stop at the adjacent peptide link for the cation conformation. These results stress the probable influence of the electronic coupling between the states rather than the existence of a barrier on the charge pathway to explain the difference between the peptides in the charge-transfer process leading to the loss of an iminium [NH(2)=CHR](+) cation. The dissociation energy of the asymptote related to the formation of this NH(2) terminus iminium cation was calculated for few species and it appears that the excess energy available for dissociation is significant when starting from the lowest energy conformations of the neutral or the cation, provided that the charge transfer is effective. It was also found that the amino acids did not conserve their energetic properties and their zero order energy levels turned to a complete new energetic scheme corresponding to the conformation of the peptide.     

Complexation of silver and co-recovered metals with novel aza-crown ether macrocycles by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is used to evaluate the metal binding selectivities of an array of novel caged macrocycles for silver, gold, copper, nickel, zinc, iron, lead, manganese and alkali metal ions. It is found that five of the new compounds display silver selectivity, and their relative affinities for various metals depend on the type, number, and arrangement of heteroatoms (N, O), the cavity size, and the presence of aromatic substituents. Alkali metal cation binding studies are used to evaluate the size-selectivities of the cavities of the macrocycles. Electronic structure calculation by B3LYP density function theory methods were used to model the metal complexes. The presence of nitrogen atoms in the macrocyclic ring is essential for silver selectivity over other transition metals and alkali metal ions, and the presence of aromatic groups also enhances silver avidity. Macrocycle 3, a triaza-18-crown-6 analog modified with two phenyl groups and a cage group, is capable of selective extraction of Ag+ from aqueous solutions in the presence of other transition metal ions and the most common alkali and alkaline earth metal ions.     

Specificity in the Cu interactions with prion protein fragments and related His-rich peptides from mammals to fishes

The prion proteins may play a critical role in copper homeostasis and the antioxidant activity in the brain. This review presents the state of art in the studies on Cu²⁺ prion systems. The proteins discussed are from different species from mammals to fishes. All proteins are His-rich and the research discussed clearly indicates the basic role of imidazole side chains and the adjacent amide nitrogen atoms in metal ion binding. Prions represent the family of proteins with new mode of Cu²⁺ binding which includes the amide nitrogen coordination. The multi-imidazole coordination is also likely and it can play a critical role in the antioxidant activity of the copper–prion complexes. The combination of the imidazole and amide nitrogen atoms to Cu²⁺ ions could also be relevant in histidine-rich peptide antibiotics including demegen. The impact of peptide sequence and His positions on copper binding ability is also discussed.     

Determination of alkali metal ion binding selectivities of calixarenes by matrix-assisted laser desorption ionization and electrospray ionization in a quadrupole ion trap

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) are used to evaluate the alkali metal ion binding selectivities of a series of calixarenes. Each calixarene of interest is mixed with one or more alkali metal salts (1:100 ratio of calixarene to metal), either in the ESI solution or on the MALDI probe surface, and the relative binding selectivities are directly determined from the intensities of the calixarene/metal complexes in the mass spectra. For t-butylcalix[4]arene-tetraacetic acid tetraethyl ester (calixarene 1), complexation of Na⁺ is favored over complexation of K⁺, in agreement with prior solution results obtained by conventional methods. For the three calixarenes that do not have t-butyl groups on the upper rims, the calixarenes preferentially bind K⁺ over Na⁺, thus demonstrating that size selective complexation can be probed with both the ESI and MALDI methods. Collision-activated dissociation results indicate that the phenyl oxygens, but not necessarily the ethoxy ethyl oxygens of the lower rims, are the primary binding sites for the alkali metal ions.     

Theoretical investigations on geometrical and electronic structures of silver clusters

Geometrical structures and ground and excited states of silver clusters are theoretically investigated using long-range corrected (LC) density functional theory (DFT) calculations. The discrepancy between HOMO-LUMO gaps and the vertical ionization potential minus vertical electron affinity values, which should be the same values, is explored to reveal the significance of long-range exchange effects on the electronic states of metal clusters. The difference between HOMO-LUMO gaps and HOMO-LUMO excitation energies, which is called “exciton binding energy,” is also tested. As a result, it is found that the long-range exchange effects are requisite in DFT calculations to quantitatively investigate the ground and excited states of metal clusters. © 2018 Wiley Periodicals, Inc.     

Gas-phase investigation of Pd(II)-alanine complexes with small native and derivatized peptides containing histidine

We report here the generation of gas-phase complexes containing Pd(II), a ligand (deprotonated alanine, A-), and/or N-terminus derivatized peptides containing histidine as one of the amino acids. The species were produced by electrospray ionization, and their gas-phase reactions were investigated using ion-trap tandem mass spectrometry. Pd(II) forms a stable diaqua complex in the gas phase of the formula, [Pd(A-) (H(2)O)(2)]+, (where A- = deprotonated alanine) along with ternary complexes containing A- and peptide. The collision-induced dissociation (CID) patterns of the binary and ternary complexes were investigated, and the dissociation patterns for the ternary complexes suggest that: (a) the imidazole ring of the histidine side group may be the intrinsic binding site of the metal ion, and (b) the peptides fragment primarily by cleavage of the amide bond to the C-terminal side of the histidine residues. These observations are in accord with previous solution-state studies in which Pd(II) was shown to cause hydrolysis of an amide bond of a peptide at the same position.     

Complexes of tetracyclines with divalent metal cations investigated by stationary and femtosecond-pulsed techniques

Spectroscopic techniques both in steady-state (in absorption and emission) and pulsed (absorption of excited states with femtosecond resolution) conditions were used to study the complexation process between six molecules belonging to the tetracycline family and Mg(2+); in the case of TC the study was extended to the metal ions Ca(2+) and Cu(2+). The study was carried out in aqueous solution at various pH values, where one acid-base form of the substrate prevails over the others. The processing of experimental results, performed by means of Singular Value Decomposition and Global Analysis methods, allowed us to evaluate the extent of interaction through the association constants, to identify the number of equilibria present in solution and the stoichiometry (1:1 or 1:2) of the tetracycline:metal ion complex, and to define the spectral and photophysical properties of the latter (in terms of fluorescence quantum yields, lifetimes and rate constants). In fact, the (allowed) radiative decay process is a minor root for the lowest excited state of the complexes which mainly decay to the ground state by internal conversion. Details of the complexation sites are proposed for the various protonated forms of tetracyclines, and for the various cations in the case of TC. In particular, the molecular structure seems to affect significantly the dynamics of interaction when the upper peripheral region of tetracycline is rich in additional hydroxyl groups. Moreover, the state of protonation of the substrate produces changes in the order of the complexation sites, whose affinity for the cation increases significantly when they are negatively charged owing to the loss of protons. Magnesium and calcium (hard cations) give similar interactions, at least in acid solution, while copper(ii) (borderline cation) binds more efficiently on different sites, thus forming complexes with different properties.     

Insight into indole interactions from alkali metal chloride effects on a tryptophan zipper beta-hairpin peptide

Weakly solvated, low charge density, alkali metal cations (K+ and Rb+) destabilize tryptophan zipper (trpzip) peptides with an effectiveness (for Rb+) similar to that of the protein denaturant urea. An analysis of alkali metal cation effects on polypeptides stabilized predominantly either by hydrogen bonds or by the classical hydrophobic effect indicates that the alkali metals attenuate stabilizing interactions involving the tryptophan indole groups. Destabilization does not result from electrolyte screening of the electrostatic component of the indole-indole interaction, but is likely to involve direct interaction of the low charge density cation with the indole group in a cation-pi interaction. The observations highlight a general simplicity in the nature of molecular interactions in solution, in which stabilizing contributions to polypeptide and protein structures are attenuated by solutes of a complementary nature.     

Structural and dynamic characterization of copper(II) binding of the human prion protein outside the octarepeat region

Human prion protein (hPrP) fragments encompassing the 91-120 region, namely hPrP92-100 (SP1), hPrP106-113 (SP2), hPrP91-120 (LP1), and hPrP91-114 (LP2), were considered for delineation of the Cu(II)-binding site(s). NMR and EPR spectroscopy results obtained from LP1 or LP2 were compared with those obtained from SP1 and SP2. The coexistence of two binding sites, one centered at His96 and the other at His111, was evidenced and ratified by ESI mass spectrometry at low and high metal:peptide ratios. While room-temperature NMR spectroscopy data were consistent with the binding site centered on His111 being approximately fourfold stronger than that centered on His96, low-temperature EPR spectroscopy results yielded evidence for the opposite trend. This disagreement, which has also occurred in the literature, was clarified by temperature-dependent molecular dynamics runs that demonstrated Met112 approaching the metal at room temperature, a process that is expected to stabilize the His111-centered binding site through hydrophobic shielding of the metal coordination sphere.     

Influence of the water molecules (n?=?1–6) on the interaction between Li+, Na+, K+ cations and indole molecule as tryptophan amino acid residue

Influence of the addition of water molecules (n = 1–6) on the interaction energy between Li⁺, Na⁺, K⁺ cations and indole molecule as tryptophan amino acid residue is considered at MP2(FULL)/6-311++G(d,p)//B3LYP/6-311++G(d,p) levels of theory. The calculations suggest that the size of cation and the number of water molecules are two important factors that affect the interaction energy between the hydrated metal cation and indole molecule. The strength of cation–π interactions get substantially reduced when the metal ion is solvated or the size of metal cation increases. Quantum theory of atoms in molecules analysis of cation–π interaction indicates that there is a correlation between the electron density (ρ(r)) in the cage critical points generated upon complexation and the distance between metal cation and centroid of phenyl ring in indole molecule.     

Metalated peptide fibers derived from a natural metal-binding peptide motif

Biological molecules serve as convenient scaffolds for the construction of nanoscopic architectures which can effectively interact with small molecules and metal complexes to extend their scope for nano(bio)technological applications. Metalloproteins possess natural metal ion binding motifs and the possibility of using these sequences to generate metalated peptide conjugates with defined metal ion coordination offers a facile entry into metalated supramolecular aggregates. This report describes the formation of metalated fibers from Cu-binding octarepeat motifs of the prion protein. Conjugate 1 effectively binds copper, silver, and manganese, leading to persistent length and thermally stable peptide fibers, which could be applied for molecular bioelectronic applications.     

Where does the electron go? Electron distribution and reactivity of peptide cation radicals formed by electron transfer in the gas phase

We report the first detailed analysis at correlated levels of ab initio theory of experimentally studied peptide cations undergoing charge reduction by collisional electron transfer and competitive dissociations by loss of H atoms, ammonia, and N-C alpha bond cleavage in the gas phase. Doubly protonated Gly-Lys, (GK + 2H) (2+), and Lys-Lys, (KK + 2H) (2+), are each calculated to exist as two major conformers in the gas phase. Electron transfer to conformers with an extended lysine chain triggers highly exothermic dissociation by loss of ammonia from the Gly residue, which occurs from the ground ( X ) electronic state of the cation radical. Loss of Lys ammonium H atoms is predicted to occur from the first excited ( A ) state of the charge-reduced ions. The X and A states are nearly degenerate and show extensive delocalization of unpaired electron density over spatially remote groups. This delocalization indicates that the captured electron cannot be assigned to reduce a particular charged group in the peptide cation and that superposition of remote local Rydberg-like orbitals plays a critical role in affecting the cation-radical reactivity. Electron attachment to ion conformers with carboxyl-solvated Lys ammonium groups results in spontaneous isomerization by proton-coupled electron transfer to the carboxyl group forming dihydroxymethyl radical intermediates. This directs the peptide dissociation toward NC alpha bond cleavage that can proceed by multiple mechanisms involving reversible proton migrations in the reactants or ion-molecule complexes. The experimentally observed formations of Lys z (+*) fragments from (GK + 2H) (2+) and Lys c (+) fragments from (KK + 2H) (2+) correlate with the product thermochemistry but are independent of charge distribution in the transition states for NC alpha bond cleavage. This emphasizes the role of ion-molecule complexes in affecting the charge distribution between backbone fragments produced upon electron transfer or capture.     

A Doppel α‐Helix Peptide Fragment Mimics the Copper(II) Interactions with the Whole Protein

The doppel protein (Dpl) is the first homologue of the prion protein (PrP^C) to be discovered; it is overexpressed in transgenic mice that lack the prion gene, resulting in neurotoxicity. The whole prion protein is able to inhibit Dpl neurotoxicity, and its N‐terminal domain is the determinant part of the protein function. This region represents the main copper(II) binding site of PrP^C. Dpl is able to bind at least one copper ion, and the specific metal‐binding site has been identified as the histidine residue at the beginning of the third helical region. However, a reliable characterization of copper(II) coordination features has not been reported. In a previous paper, we studied the copper(II) interaction with a peptide that encompasses only the loop region potentially involved in metal binding. Nevertheless, we did not find a complete match between the EPR spectroscopic parameters of the copper(II) complexes formed with the synthesized peptide and those reported for the copper(II) binding sites of the whole protein. Herein, the synthesis of the human Dpl peptide fragment hDpl(122–139) (Ac‐KPDNKLHQQVLWRLVQEL‐NH₂) and its copper(II) complex species are reported. This peptide encompasses the third α helix and part of the loop linking the second and the third helix of human doppel protein. The single‐point‐mutated peptide, hDpl(122–139)D124N, in which aspartate 124 replaces an asparagine residue, was also synthesized. This peptide was used to highlight the role of the carboxylate group on both the conformation preference of the Dpl fragment and its copper(II) coordination features. NMR spectroscopic measurements show that the hDpl(122–139) peptide fragment is in the prevailing α‐helix conformation. It is localized within the 127–137 amino acid residue region that represents a reliable conformational mimic of the related protein domain. A comparison with the single‐point‐mutated hDpl(122–139)D124N reveals the significant role played by the aspartic residue in addressing the peptide conformation towards a helical structure. It is further confirmed by CD measurements. Potentiometric titrations were carried out in aqueous solutions to obtain the stability constant values of the species formed by copper(II) with the hDpl peptides. Spectroscopic studies (EPR, NMR, CD, UV/Vis) were performed to characterize the coordination environments of the different metal complexes. The EPR parameters of the copper(II) complexes with hDpl(122–139) match those of the previously reported copper(II) binding sites of the whole hDpl. Addition of the copper(II) ion to the peptide fragment does not alter the helical conformation of hDpl(122–139), as shown by CD spectra in the far‐UV region. The aspartate‐driven preorganized secondary structure is not significantly modified by the involvement of Asp124 in the copper(II) complex species that form in the physiological pH range. To elaborate on the potential role of copper(II) in the recently reported interaction between the PrP^C and Dpl, the affinity of the copper(II) complexes towards the prion N terminus domain and the binding site of Dpl was reported.     

Toward a general mechanism of electron capture dissociation

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 N-Calpha bond dissociation and transition state energies in charge-stabilized NMA anions are 20-50 kJ mol(-1) greater than in the hydrogen atom adduct. The zwitterions formed by electron capture have proton affinities that were calculated as 1030-1350 kJ mol(-1), 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 N-Calpha bond cleavage. This mechanism explains the low selectivity of N-Calpha 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 N-H bond dissociation in weakly bound ground electronic states of hypervalent ammonium radicals, as represented by methylammonium, CH3NH3*, but has a negligible effect on the N-H 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.     

Interaction of the human prion PrP(106-126) sequence with copper(II), manganese(II), and zinc(II): NMR and EPR studies

The synthetic peptide encompassing residues 106-126 (PrP106-126, KTNMKHMAGAAAAGAVVGGLG) of the human prion protein was considered for its binding properties toward copper(II), manganese(II) and zinc(II) at pH 5.7. 1H and 13C 1D spectra, 1H spin-lattice relaxation rates, and 1H-15N and 1H-13C HSQC 2D experiments were obtained in the absence and in the presence of metal ions. While Zn(II) was found to yield negligible effects upon any NMR parameter, metal-peptide association was demonstrated by the paramagnetic effects of Cu(II) and Mn(II) upon 1D and 2D spectra. Delineation of structures of metal complexes was sought by interpreting the paramagnetic effect on 1H spin-lattice relaxation rates. Exchange of peptide molecules from the metal coordination sphere was shown to provide sizable contribution to the observed relaxation rates. Such contribution was calculated in the case of Cu(II); whereas the faster paramagnetic rates of peptide molecules bound to Mn(II) were determining spin-lattice relaxation rates almost exclusively dominated by exchange. Proton-metal distances were therefore evaluated in the case of the Cu(II) complex only and used as restraints in molecular dynamics calculations where from the structure of the complex was obtained. The peptide was shown to bind copper through the imidazole nitrogen and the ionized amide nitrogen of His-111 and the amino-terminal group with the terminal carboxyl stabilizing the coordination sphere through ionic interactions. The data were interpreted as to demonstrate that the hydrophobic C-terminal region was not affecting the copper-binding properties of the peptide and that this hydrophobic tail is left free to interact with other target molecules. As for the complex with Mn(II), qualitative information was obtained on carbonyl oxygens of Gly-124 and Leu-125, beyond the terminal Gly-126 carboxyl, being at close distance from the metal ion, that also interacts, most likely, through a hydrogen bond of metal-bound water, with the imidazole ring of His-111.     

Ground and excited state resonance Raman spectra of an azacrown-substituted [(bpy)Re(CO)3L]+ complex: characterization of excited states, determination of structure and bonding, and observation of metal cation release from the azacrown

A [(bpy)Re(CO)3L+] complex (bpy = 2,2'-bipyridine) in which L contains a phenyl-azacrown ether that is attached to Re via an amidopyridyl linking group has been studied by steady state and nanosecond time-resolved resonance Raman spectroscopy. Vibrational band assignments have been aided by studies of model complexes in which a similar electron-donating dimethylamino group replaces the azacrown or in which an electron-donor group is absent, and by density functional theory calculations. The ground state resonance Raman spectra show nu(bpy) and nu(CO) bands of the (bpy)Re(CO)3 group when excitation is exclusively in resonance with the Re --> bpy metal-to-ligand charge-transfer (MLCT) transition, whereas L ligand bands are dominant when it is in resonance with the strong intra-ligand charge-transfer (ILCT) transition present for L ligands with electron-donor groups. Transient resonance Raman (RR) spectra obtained on single color (385 nm) pulsed excitation of the complexes in which an electron-donor group is absent show bpy*- bands of the MLCT excited state, whereas those of the complexes with electron-donor groups show both bpy*- bands and a down-shifted nu(CO) band that together are characteristic of an L-to-bpy ligand-to-ligand charge-transfer (LLCT) excited state. Samples in which a metal cation (Li+, Na+, Ca2+, Ba2+) is bound to the azacrown in the ground state show bands from both excited states, consistent with a mechanism in which the LLCT state forms after metal cation release from the MLCT state. Nanosecond time-resolved RR spectra from two-color (355 nm pump, 500 nm probe) experiments on the electron-donor systems show L-ligand bands characteristic of the LLCT state; the same bands are observed from samples in which a metal cation is bound to the azacrown in the ground state, and their time dependence is consistent with the proposed mechanism in which the rate constant for ion release in the MLCT state depends on the identity of the metal cation.     

The effect of metal cation binding on the protein, lipid and retinal isomeric ratio in regenerated bacteriorhodopsin of purple membrane

The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800-1000 cm-1 are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the alpha 1-helix region of bR, converting the portion of its alpha 1-helical domain into beta-turn or disordered coil. In addition, the influence of Ho3+ binding on the protein and lipid is observed to be larger than that of Ca2+. These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO- groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.     

Site specific interaction between ZnO nanoparticles and tryptophan: a first principles quantum mechanical study

First principles density functional theory calculations are performed on tryptophan-ZnO nanoparticles complex in order to study site specific interactions between tryptophan and ZnO. The calculated results find the salt bridge structure involving the -COOH group and ZnO cluster to be energetically more favorable than other interacting sites, such as indole and amine groups in tryptophan. The interaction between tryptophan and ZnO appears to be mediated by both ionic and hydrogen bonds. The calculated molecular orbital energy levels and charge distributions suggest non-radiative energy transfer from an excited state of tryptophan to states associated with ZnO, which may lead to a reduction in the emission intensity assigned to the π-π* transition of the indole functional group of tryptophan.     

Determination of the glycosylation site of flavonoid monoglucosides by metal complexation and tandem mass spectrometry

Metal complexation and tandem mass spectrometry were used to differentiate C- and O-bonded flavonoid monoglucoside isomers. Electrospray ionization of solutions containing a flavonoid glycoside and a metal salt led to the generation of the key [M(II) (L) (L-H)](+) complexes, where M is the metal ion and L is the flavonoid glycoside. Thirteen flavonoid monoglucosides were examined in combination with Ca(II), Mg(II), Co(II), Ni(II), and Cu(II). Collisional activated dissociation (CAD) of the [M(II) (L) (L-H)](+) complexes resulted in diagnostic mass spectra, in contrast to the CAD mass spectra of the protonated, deprotonated, and sodium-cationized flavonoid glucosides. Five common sites of glycosylation could be predicted based on the fragmentation patterns of the flavonoid glucoside/magnesium complexes, while flavonoid glucoside/calcium complexes also were effective for location of the glycosylation site when MS(3) was employed. Cobalt, nickel and copper complexation had only limited success in this application. The metal complexation methods were also applied for characterization of a flavonoid rhamnoside, and the dissociation pathways of the metal complexes indicate that flavonoid rhamnosides have distinctive dissociation features from flavonoid glucosides.