Complementary Ion Research Articles

Sequestration of the ionizing proton in singly charged metastable protein ions generated by MALDI

A hypothesis is proposed for the preferential ionization/protonation of arginine residues in metastable protein ions. This phenomenon has significant consequences for detection of protein fragments resulting from polypeptide backbone cleavage. Preferential ionization at arginine (when present) is based upon MS/MS analysis of singly charged/protonated metastable protein ions: YahO, CspC, CspE, B-subunit of Shiga toxin 2 and thioredoxin, that have either zero or one arginine residue in their sequences. For protein ions having no arginine, we observe multiple b/y complementary fragment ion pairs (CFIP) by MALDI-MS/MS post-source decay demonstrating that the ionizing proton is located at multiple locations amongst the packet of protein precursor ions being analyzed. By contrast, for protein ions having one arginine, we observe little or no CFIP, and almost every fragment ion has the arginine residue in its sequence. This preferential proton sequestration is presumably due to the higher gas phase basicity of arginine compared to lysine, although lysine residues are often more numerous than arginine residues. As this phenomenon has been noted previously in the ionization of tryptic peptides by MALDI where the basic residue (K or R) is located exclusively at the C-terminus (if there are no missed proteolytic cleavages), we conclude that rapid proton transfer in the gas phase occurs via protein/matrix collisions in the MALDI plume. Upon proton transfer to the side-chain of an arginine residue, the proton is effectively immobilized by its guanidino functional group. An alternative explanation is proton exchange from the ionized side-chain of a lysine to the side-chain of the arginine in the metastable ion.

Electrostatic Potential Field Effects on Amine Macrocyclizations within Yoctoliter Spaces: Supramolecular Electron Withdrawing/Donating Groups.

The central role of Coulombic interactions in enzyme catalysis has inspired multiple approaches to sculpting electrostatic potential fields (EPFs) for controlling chemical reactivity, including ion gradients in water microdroplets, the tips of STMs, and precisely engineered crystals. These are powerful tools because EPFs can affect all reactions, even those whose mechanisms do not involve formal charges. For some time now, supramolecular chemists have become increasingly proficient in using encapsulation to control stoichiometric and catalytic reactions. However, the field has not taken advantage of the broad range of nanocontainers available to systematically explore how EPFs can affect reactions within their inner-spaces. With that idea in mind, previously, we reported on how positively and negatively charged supramolecular capsules can modulate the acidity and reactivity of thiol guests bound within their inner, yoctoliter spaces (Cai, X.; Kataria, R.; Gibb, B. C. J. Am. Chem. Soc. 2020, 142, 8291–8298; Wang, K.; Cai, X.; Yao, W.; Tang, D.; Kataria, R.; Ashbaugh, H. S.; Byers, L. D.; Gibb, B. C. J. Am. Chem. Soc.2019, 141, 6740–6747). Building on this, we report here on the cyclization of 14-bromotetradecan-1-amine inside these yoctoliter containers. We examine the rate and activation thermodynamics of cyclization (Eyring analysis), both in the absence and presence of exogenous salts whose complementary ion can bind to the outside of the capsule and hence attenuate its EPF. We find the cyclization rates and activation thermodynamics in the two capsules to be similar, but that for either capsule attenuation of the EPF slows the reaction down considerably. We conclude the capsules behave in a manner akin to covalently attached electron donating/withdrawing groups in a substrate, with each capsule enforcing their own deviations from the idealized SN2 mechanism by moving electron density and charge in the activated complex and TS, and that the idealized SN2 mechanism inside the theoretical neutral host is relatively difficult because of the lack of solvation of the TS.

LIBS applicability for investigation of re-deposition and fuel retention in tungsten coatings exposed to pure and nitrogen-mixed deuterium plasmas of Magnum-PSI

We have investigated the applicability of Laser Induced Breakdown Spectroscopy (LIBS) for analyzing the changes in the composition and fuel retention of W and W-Ta coatings following exposure to D2 or mixed D2-N2 plasma beams in the linear plasma device Magnum PSI. The exposed samples were characterized by in situ ns-LIBS and complementary analysis methods Secondary Ion Mass Spectroscopy, Energy Dispersive x-ray spectroscopy and Nuclear Reaction Analysis. In agreement with the used complementary analysis methods, LIBS revealed the formation of up to 400 nm thick co-deposited surface layer in the central region of the coatings which contained a higher concentration of the main plasma impurities, such as N, and metals, such as Ta and Mo, the latter originating mainly from the substrate and from the plasma source. The deuterium retention on the other hand was highest outside from the central region of the coatings.

Charge transfer dissociation of a branched glycan with alkali and alkaline earth metal adducts.

Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He-CTD) and low-energy collision-induced dissociation (LE-CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H+ , Na+ , K+ , Ca2+ , and Mg2+ . Regardless of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching patterns. In contrast, the LE-CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE-CID of [M + K + H]2+ and [M + Na]+ precursors generated a few cross-ring cleavages, but they were not sufficient to identify the 1,4-linkage and 1,6-branching pattern of the XXXG xyloglucan. He-CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE-CID and He-CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He-CTD, the [M + Mg]2+ precursor generated more singly charged product ions than [M + Ca]2+ , either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He-CTD of the [M + 2Na]2+ and the [M + 2K]2+ precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross-ring cleavages observed.

Geophysically based analysis of breakthrough curves and ion exchange processes in soil

Abstract. Breakthrough curves (BTCs) are a valuable tool for qualitative and quantitative examination of transport patterns in porous media. Although breakthrough (BT) experiments are simple, they often require extensive sampling and multi-component chemical analysis. In this work, we examine spectral induced polarization (SIP) signals measured along a soil column during BT experiments in homogeneous and heterogeneous soil profiles. Soil profiles were equilibrated with an NaCl background solution, and then a constant flow of either CaCl2 or ZnCl2 solution was applied. The SIP signature was recorded, and complementary ion analysis was performed on the collected outflow samples. Our results confirm that changes to the pore-water composition, ion exchange processes and profile heterogeneity are detectable by SIP: the real part of the SIP-based BTCs clearly indicated the BT of the non-reactive ions as well as the retarded BT of cations. The imaginary part of the SIP-based curves changed in response to the alteration of ion mobility around the electrical double layer (EDL) and indicated the initiation and the termination of the cation exchange reaction. Finally, both the real and imaginary components of the complex conductivity changed in response to the presence of a coarser textured layer in the heterogeneous profile.

TMTpro Complementary Ion Quantification Increases Plexing and Sensitivity for Accurate Multiplexed Proteomics at the MS2 Level.

Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but accurate quantification of relative protein changes is impaired by interference from co-isolated peptides. Interference can be reduced by using MS3-based quantification, but this reduces sensitivity and requires specialized instrumentation. An alternative approach is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. However, complementary ions of the popular TMT-tag form inefficiently and multiplexing is limited to five channels. Here, we evaluate and optimize complementary ion quantification for the recently released TMTpro-tag, which increases complementary ion plexing capacity to eight channels (TMTproC). Furthermore, the beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC, resulting in ∼65% more proteins quantified compared to TMTpro-MS3 and ∼18% more when compared to real-time-search TMTpro-MS3 (RTS-SPS-MS3). TMTproC quantification is more accurate than TMTpro-MS2 and even superior to RTS-SPS-MS3. We provide the software for quantifying TMTproC data as an executable that is compatible with the MaxQuant analysis pipeline. Thus, TMTproC advances multiplexed proteomics data quality and widens access to accurate multiplexed proteomics beyond laboratories with MS3-capable instrumentation.

Analysis of Peptide Stereochemistry in Single Cells by Capillary Electrophoresis-Trapped Ion Mobility Spectrometry Mass Spectrometry.

Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell-cell signaling peptides. The multiplicity of endogenous cell-cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE-TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.

Characterization of historical minai and colorful luster glaze ceramics, using ionoluminescence technique

Some historical minai and colorful luster glaze ceramic samples were investigated by means of ionoluminescence technique. The complementary ion beam analysis techniques RBS and PIXE were employed to reveal their elemental compositions and depth profiles. The samples were discovered in the historical site of Tappeh Ghale in Iran, which dates back to 12th century. The ionoluminescence spectra of the samples reveal the strong dependence of the luminescence bands, mostly originating from oxygen-deficient centers, on the elemental compositions of the glaze samples. It is inferred that most of the luminescence centers likely have been created during the formation process of tin-opacified lead-rich glazes. The demonstrated capabilities of ionoluminescence in the current work, confirm the possibility of its application for fast and non-destructive characterization of historical glaze samples.

Exploration of Matrix Effects in Laser Ablation Inductively Coupled Plasma Mass Spectrometry Imaging of Cisplatin-Treated Tumors.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

Synthesis and in-situ characterization of photochromic yttrium oxyhydride grown by reactive e−-beam evaporation

We report on controlled growth of photochromic yttrium oxyhydride thin films monitored by in-situ composition depth profiling. Films were grown by reactive e−-beam evaporation and subsequently oxidized, while simultaneously tracking the oxygen and hydrogen concentrations. Sample composition and photochromic response were characterized in-situ using non-destructive ion beam analysis and image analysis, respectively – as well as complementary ex-situ ion beam methods, X-ray diffraction and optical spectrophotometry. We show that photochromic yttrium oxyhydride can be grown as yttrium dihydride, which is then oxidized to O/H ratios triggering the photochromic response.

Cross-sectional characterization of the conversion layer formed on AA2024-T3 by a lithium-leaching coating

This work focuses on the cross-sectional characterization of the protective conversion layer formed on AA2024-T3 by lithium-leaching from a polyurethane coating in a corrosive environment. The layer shows a multi-layered arrangement comprising nanoscopic local phases. Transmission electron microscopy (TEM) and complementary high-resolution secondary ion mass spectroscopy (SIMS) were employed to observe the cross-sections of the entire layer formed at different locations of a 1-mm-wide scribe, in terms of morphology, structure and chemical composition. The conversion layer was comprised of two ubiquitous sublayers; a thin dense layer (i.e. 150 nm) adjacent the alloy substrate and a porous layer. The former represents an amorphous lithium-containing pseudoboehmite phase, Li-pseudoboehmite, whereas the latter is composed of amorphous and crystalline products; an outer columnar layer merely seen on the peripheral region is also crystalline. Through a sandwich structure and the d(003) basal spacing, the crystalline phases were identified as Li-Al layered double hydroxide. Although lithium was found uniformly spread within different regions, the local phases with no/low concentration of lithium were revealed with energy filtered TEM and confirmed with SIMS analysis.

Top-Down Characterization of Denatured Proteins and Native Protein Complexes Using Electron Capture Dissociation Implemented within a Modified Ion Mobility-Mass Spectrometer.

Electron-based fragmentation methods have revolutionized biomolecular mass spectrometry, in particular native and top-down protein analysis. Here, we report the use of a new electromagnetostatic cell to perform electron capture dissociation (ECD) within a quadrupole/ion mobility/time-of-flight mass spectrometer. This cell was installed between the ion mobility and time-of-flight regions of the instrument, and fragmentation was fast enough to be compatible with mobility separation. The instrument was already fitted with electron transfer dissociation (ETD) between the quadrupole and mobility regions prior to modification. We show excellent fragmentation efficiency for denatured peptides and proteins without the need to trap ions in the gas phase. Additionally, we demonstrate native top-down backbone fragmentation of noncovalent protein complexes, leading to comparable sequence coverage to what was achieved using the instrument's existing ETD capabilities. Limited collisional ion activation of the hemoglobin tetramer before ECD was reflected in the observed fragmentation pattern, and complementary ion mobility measurements prior to ECD provided orthogonal evidence of monomer unfolding within this complex. The approach demonstrated here provides a powerful platform for both top-down proteomics and mass spectrometry-based structural biology studies.

Comparison of Fragmentation Techniques for the Structural Characterization of Singly Charged Agrochemicals.

Investigating the structure of active ingredients, such as agrochemicals and their associated metabolites, is a crucial requisite in the discovery and development of these molecules. In this study, structural characterization by electron-induced dissociation (EID) was compared to collisionally activated dissociation (CAD) on a series of agrochemicals. EID fragmentation produced a greater variety of fragment ions and complementary ion pairs leading to more complete functional group characterization compared to CAD. The results obtained displayed many more cross-ring fragmentation of the pyrimidine ring compared to the pyridine ring. Compounds that consisted of one aromatic heterocyclic moiety (azoxystrobin, fluazifop acid, fluazifop-p-butyl, and pirimiphos-methyl) displayed cross-ring fragmentation while compounds with only aromatic hydrocarbon rings (fenpropidin and S-metolachlor) displayed no cross-ring fragmentation. The advantages of high-resolution accurate mass spectrometry (HRAM MS) are shown with the majority of assignments at ppb range error values and the ability to differentiate ions with the same nominal mass but different elemental composition. This highlights the potential for HRAM MS and EID to be used as a tool for structural characterization of small molecules with a wide variety of functional groups and structural motifs.

Dissociative electron transfer of copper(ii) complexes of glycyl(glycyl/alanyl)tryptophan in vacuo: IRMPD action spectroscopy provides evidence of transition from zwitterionic to non-zwitterionic peptide structures.

We report herein the first detailed study of the mechanism of redox reactions occurring during the gas-phase dissociative electron transfer of prototypical ternary [CuII(dien)M]˙2+ complexes (M, peptide). The two final products are (i) the oxidized non-zwitterionic π-centered [M]˙+ species with both the charge and spin densities delocalized over the indole ring of the tryptophan residue and with a C-terminal COOH group intact, and (ii) the complementary ion [CuI(dien)]+. Infrared multiple photon dissociation (IRMPD) action spectroscopy and low-energy collision-induced dissociation (CID) experiments, in conjunction with density functional theory (DFT) calculations, revealed the structural details of the mass-isolated precursor and product cations. Our experimental and theoretical results indicate that the doubly positively charged precursor [CuII(dien)M]˙2+ features electrostatic coordination through the anionic carboxylate end of the zwitterionic M moiety. An additional interaction exists between the indole ring of the tryptophan residue and one of the primary amino groups of the dien ligand; the DFT calculations provided the structures of the precursor ion, intermediates, and products, and enabled us to keep track of the locations of the charge and unpaired electron. The dissociative one-electron transfer reaction is initiated by a gradual transition of the M tripeptide from the zwitterionic form in [CuII(dien)M]˙2+ to the non-zwitterionic M intermediate, through a cascade of conformational changes and proton transfers. In the next step, the highest energy intermediate is formed; here, the copper center is 5-coordinate with coordination from both the carboxylic acid group and the indole ring. A subsequent switch back to 4-coordination to an intermediate IM1, where attachment to GGW occurs through the indole ring only, creates the structure that ultimately undergoes dissociation.

A simulation study of the influence of the traveling wave patterns on ion mobility separations in structures for lossless ion manipulations.

Probing molecular properties in the gas phase requires the integration of complementary ion manipulation approaches such as ion mobility spectrometry. Structures for lossless ion manipulations (SLIM) have recently been developed to perform ultra-high resolution ion mobility separations using traveling waves as well as providing other advanced capabilities. Despite its success, the design aspects of SLIM have not been fully explored and remained largely unchanged. Here, we report on a computational study using SIMION simulations of a number of traveling wave (TW) patterns that can be used in SLIM. The TW pattern used in the current SLIM device is a set of 8 electrodes where, at any time, 4 electrodes are held at high voltage (i.e., 1111), while the other 4 electrodes are held at low voltage (i.e., 0000), forming one micro-trapping region of 11110000 pattern. Ion trajectory simulations demonstrated the feasibility to simplify the 8-electrode set to a shorter pattern (e.g., 6-electrode or 4-electrode set) while maintaining or improving the performance. The RF and TW amplitudes, guard voltage, and TW speed were optimized subsequently on the symmetric patterns of the 4-, 6-, and 8-electrode sets to further improve the performance. The resolution, peak broadening, peak capacity, and peak generation rate of each pattern were evaluated, showing that the 111000 pattern of the 6-electrode set has comparable performance to the current 11110000 pattern and is always better than the 1100 pattern. This work provides insight into the feasibility for simplification and modification of the TW configuration in SLIM and other traveling wave devices.

Multi-scheme chemical ionization inlet (MION) for fast switching of reagent ion chemistry in atmospheric pressure chemical ionization mass spectrometry (CIMS) applications

Abstract. A novel chemical ionization inlet named the Multi-scheme chemical IONization inlet (MION), Karsa Ltd., Helsinki, Finland) capable of fast switching between multiple reagent ion schemes is presented, and its performance is demonstrated by measuring several known oxidation products from much-studied cyclohexene and α-pinene ozonolysis systems by applying consecutive bromide (Br−) and nitrate (NO3-) chemical ionization. Experiments were performed in flow tube reactors under atmospheric pressure and room temperature (22 ∘C) utilizing an atmospheric pressure interface time-of-flight mass spectrometer (APi-ToF-MS, Tofwerk Ltd., Thun, Switzerland) as the detector. The application of complementary ion modes in probing the same steady-state reaction mixture enabled a far more complete picture of the detailed autoxidation process; the HO2 radical and the least-oxidized reaction products were retrieved with Br− ionization, whereas the highest-oxidized reaction products were detected in the NO3- mode, directly providing information on the first steps and on the ultimate endpoint of oxidation, respectively. While chemical ionization inlets with multiple reagent ion capabilities have been reported previously, an application in which the charging of the sample occurs at atmospheric pressure with practically no sample pretreatment, and with the potential to switch the reagent ion scheme within a second timescale, has not been introduced previously. Also, the ability of bromide ionization to detect highly oxygenated organic molecules (HOM) from atmospheric autoxidation reactions has not been demonstrated prior to this investigation.

Methionine oxidation in α-synuclein inhibits its propensity for ordered secondary structure

α-Synuclein (AS) is an intrinsically disordered protein highly expressed in dopaminergic neurons. Its amyloid aggregates are the major component of Lewy bodies, a hallmark of Parkinson's disease (PD). AS is particularly exposed to oxidation of its methionine residues, both in vivo and in vitro Oxidative stress has been implicated in PD and oxidized α-synuclein has been shown to assemble into soluble, toxic oligomers, rather than amyloid fibrils. However, the structural effects of methionine oxidation are still poorly understood. In this work, oxidized AS was obtained by prolonged incubations with dopamine (DA) or epigallocatechin-3-gallate (EGCG), two inhibitors of AS aggregation, indicating that EGCG promotes the same final oxidation product as DA. The conformational transitions of the oxidized and non-oxidized protein were monitored by complementary biophysical techniques, including MS, ion mobility (IM), CD, and FTIR spectroscopy assays. Although the two variants displayed very similar structures under conditions that stabilize highly disordered or highly ordered states, differences emerged in the intermediate points of transitions induced by organic solvents, such as trifluoroethanol (TFE) and methanol (MeOH), indicating a lower propensity of the oxidized protein for forming either α- or β-type secondary structures. Furthermore, oxidized AS displayed restricted secondary-structure transitions in response to dehydration and slightly amplified tertiary-structure transitions induced by ligand binding. This difference in susceptibility to induced folding could explain the loss of fibrillation potential observed for oxidized AS. Finally, site-specific oxidation kinetics point out a minor delay in Met-127 modification, likely due to the effects of AS intrinsic structure.

Development of a microthrust balance and ion beam measurement system: Characterizing a dual-mode thruster for spacecraft

There is a rapid progression toward spacecraft miniaturization due to the prospect of reduced launch costs, the requirement for satellite constellations, and the integration of microelectromechanical systems (MEMS). Satellite propulsion is required for increased mission flexibility and orbital debris mitigation, but down-scaling of such devices has proven non-trivial. Some electrostatic thrusters are both challenging and promising given their increase in performance with small geometry, but the relatively low thrust produced through ion acceleration limits their mission applicability. For this reason, it may be desirable to produce thrusters that are able operate across a wide range of specific impulses, depending on maneuver time, thrust bit requirements, and several other factors that influence propulsion system selection. At RAPPEL, a bimodal thruster that can operate at high specific impulse with electrospray propulsion or high thrust using cold gas expansion has been developed. Inevitably, such a propulsion system comes with complications related to performance characterization. A novel and economical data acquisition system has been established to measure the performance of both modes in high vacuum. For the cold gas mode, a torsional thrust balance was built in-house to measure thrust levels between 1−15mN, and can be easily modified for lower ranges. The natural frequency of the thrust stand was measured to be 5.6Hz. The vacuum system sustained pressures below 5mbar while the cold gas nozzle operated, and 1.3×10−5mbar during electrospray operation. A 3-D printed linear cold gas nozzle was used to validate the thrust stand. Its thrust was close to 9mN at a mass flow rate of 20mg/s of nitrogen, producing a specific impulse of just under 45s. Due to the noise present in thrust stand measurements at the sub-mN force-level produced by electrosprays, the thrust stand was built to also accommodate a complementary ion beam current measurement system. The power processing unit, used to generate square-wave ±2500V signals using optodiodes, was built to operate a bipolar ionic liquid ion source (ILIS). The rise and fall times of the push/pull switch were close to 1ms and 30ms, respectively. The electrical power system used to operate the electrospray and measure its performance is examined in detail, followed by the results from validation of both data acquisition systems.

Gold-potassium sheets intercalated into graphite: Chemistry and structure of a first stage ternary compound

A ternary graphite-potassium-gold compound K1.3Au1.5C4 was synthesized by immersing a pyrographite platelet in various potassium metal-based molten alloys. Numerous reaction parameters were investigated (duration, temperature, composition of the alloy) in order to determine the stability conditions of the ternary compound. Using complementary X-ray diffraction and ion beam analysis experiments, the optimal conditions of preparation of this compound have been pointed out. The composition and homogeneity were evidenced by ion beam analysis and X-ray diffraction allowed to determine its repeat distance (1311 pm) and its c-axis stacking sequence (K-Au-Au-Au-K). The 2D unit cell observed thanks to Transmission Electron Microscopy (TEM) is square with a = 1192 pm, no-commensurate with that of graphite.

Simple structural elucidation of ostreocin-B, a new palytoxin congener isolated from the marine dinoflagellate Ostreopsis siamensis, using complementary positive and negative ion liquid chromatography/quadrupole time-of-flight mass spectrometry.

Ostreocin-B is a new palytoxin congener of comparable complexity. Elucidation of the chemical structure by nuclear magnetic resonance has been hampered due to limited sample availability. More importantly, the dihedral angles of protons at the base of the hydroxyl groups on the ring structures are predicted to produce little couplings and thus disrupt connectivity. The present investigation solved the problem through mass spectrometry. Structural elucidation was performed by high-performance liquid chromatograph coupled to a quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source operated in positive and negative ion mode. Measurement parameters were optimized to achieve high sensitivity and a high ratio of singly charged ions. Ostreocin-D (C127 H219 N3 O53 ), another palytoxin congener possessing an unambiguously determined structure, was used as a template. The molecular formula of ostreocin-B, C127 H219 N3 O54 , indicates that it has one more oxygen atom than ostreocin-D. Comparison of the product ion spectra in negative ion mode indicates the occurrence of hydroxyl substitution at C44 in ostreocin-B, unlike in ostreocin-D. Positive ion spectra also support the 44-OH substructure by producing conjugated polyenes ascribable to the sequential loss of hydroxyls on the cyclic hemiacetal. The planar structure of ostreocin-B is assigned to 42-hydroxy-3,26-didemethyl-19-deoxypalytoxin (=44-hydroxyostreocin-D). The method used in this study is an excellent tool to obtain structural information on interspecies and intrastrain variation of palytoxin congeners in marine organisms.