Daryl Giblin

Electrospray ionization mass spectrometry and hydrogen/deuterium exchange for probing the interaction of calmodulin with calcium.

The extent of H/D exchange of the protein calmodulin in solution was monitored by mass spectrometry following electrospray ionization (ESI) of the protein. In the absence of Ca2+, approximately 115 protons are exchanged for deuteriums after 60 min. As the calmodulin is titrated with Ca2+, the extent of exchange decreases significantly (i.e., by 24 protons), indicating Ca2+-induced folding of the protein to a tighter, less solvent-accessible form. The extent of H/D exchange ceases to decrease when the amount of added Ca2+ is sufficient to convert greater than 80% of the calmodulin to a form bound by four calcium ions. Lysozyme, a protein of similar molecular weight, does not show a significant decrease in the extent of H/D exchange as it binds to Ca2+, indicating that the changes in H/D exchange for calmodulin reflect tertiary structural change that occur upon binding with Ca2+.

The Magic-Size Nanocluster (CdSe)34 as a Low-Temperature Nucleant for Cadmium Selenide Nanocrystals; Room-Temperature Growth of Crystalline Quantum Platelets

Reaction of Cd(OAc)2·2H2O and selenourea in primary-amine/secondary-amine cosolvent mixtures affords crystalline CdSe quantum platelets at room temperature. Their crystallinity is established by X-ray diffraction analysis (XRD), high-resolution transmission electron microscopy (TEM), and their sharp extinction and photoluminescence spectra. Reaction monitoring establishes the magic-size nanocluster (CdSe)34 to be a key intermediate in the growth process, which converts to CdSe quantum platelets by first-order kinetics with no induction period. The results are interpreted to indicate that the critical crystal-nucleus size for CdSe under these conditions is in the range of (CdSe)34 to (CdSe)68. The nanocluster is obtained in isolated form as [(CdSe)34(n-octylamine)16(di-n-pentylamine)2], which is proposed to function as crystal nuclei that may be stored in a bottle.

Cupric Yersiniabactin Is a Virulence-Associated Superoxide Dismutase Mimic

Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst.

Mass spectrometric analysis of pentafluorobenzyl oxime derivatives of reactive biological aldehydes

Abstract We recently demonstrated that a battery of reactive aldehydes can be generated by human neutrophils through the action of myeloperoxidase on α-amino acids. To determine the aldehydes, we formed the pentafluorobenzyloxime (PFBO) derivatives by reacting them with pentafluorobenzylhydroxylamine (PFBHA) and submitting the derivatives to gas chromatography electron-capture mass spectrometry (GC/EC/MS). Two geometric isomers are formed for each of the aldehydes, and they are separable by gas chromatography (GC) and exhibit distinguishable electron-capture (EC) mass spectra. Major fragment ions include [M − HF] · − , which probably has a six-membered ring formed via HF loss from the molecular radical anion. Subsequent decomposition of this intermediate yields other characteristic ions (e.g. those formed by elimination of NO and the anion of m / z 178). We proposed fragmentation pathways and mechanisms that are consistent with the data derived from collisionally-activated dissociations (CAD) coupled with tandem mass spectrometry, exact-mass measurements, studies of deuterium-labeled analogs, and calculations by PM3 semiemperical and ab initio methods. The generality of the structurally informative fragmentation pattern together with GC separation is the basis of a powerful means for the identification of reactive aldehydes in biological processes and the pathogenesis of disease.

High-energy collisions of Kr@C60+. with helium. Evidence for the formation of HeKr@C60+.

Abstract High energy collisions of Kr@C60+. with stationary helium atoms provide additional evidence that - as demonstrated earlier (Saunders, Jimenez-Vazquez, Cross, Mroczkowski, Gross, Giblin and Poreda) - a krypton atom penetrates the carbon network of C60-fullerenes under thermal conditions (15 atm, 600°C, 2h). In the collision experiments, signals are also observed that can best be explained in terms of a HeKr@C60+. compound.

Structural determination of oxofatty acids by charge-remote fragmentations

A strategy is described to locate the carbonyl position in oxofatty acids by utilizing charge-remote fragmentations of various molecular ions that are desorbed by fast atom bombardment (FAB). Oxofatty acids were cationized with alkali metal ions (Li+, Na+, K+, Rb+, and Cs+) to form [M+2Met−H]+ or alkaline earth metal ions (Mg2+, Ca2+, Sr2+ or Ba2+) to form [M+Met−H]+ in the gas phase. The cationized acids undergo charge-remote fragmentations upon high-energy activation, giving a product-ion pattern that has a gap corresponding to the oxo position and bordered by two high-intensity peaks. One of the peaks corresponds to an ion that is formed by the cleavage of the C-C bond β to the oxo position and proximal to the charge (β ion), whereas the other is formed from the cleavage of the C-C bond γ to the oxo position and distal to the charge (γ′ ion). The oxo position is easily determined by identifying the gap and the β and γ′ ions. Furthermore, there are two competing patterns of fragments in a CAD spectrum of an oxofatty acid or ester [M+Li]+ ion. These arise because Li+ attaches to either the oxo or the carboxylic end, as was confirmed by ab initio molecular orbital calculations. The results demonstrate that control of the fragmentation can be guided by an understanding of metal-ion affinities. Collisional activation of the anionic carboxylates gives results that are similar to those for positive ions, showing that the process is not related to the charge status. Collisional activation of [M+H]+ ions does not give structural information because the charge migrates, leading to charge-mediated fragmentations.

Mass spectrometric methods : an answer for macromolecule analysis in the 1990s

Abstract The field of mass spectrometry is now well developed for solving analytical and structural problems involving substances with molecular weights less than ca. 1000. The future challenge for mass spectrometry is in the area of macromolecule analysis and structural biology. This challenge will be met on two fronts. One is structural analysis of pieces of macromolecules, a task for tandem mass spectrometers. Tandem sector instruments offer sufficient control, reproducibility of results and ease of set-up that they will play a major role in structure studies. When designed to operate with extended array detectors, tandem sector instruments will also offer subpicomole detection limits. The second front is molecular weight measurements. Exciting advances include matrix-assisted laser desorption and electrospray ionization. The need for better means of mass analysis is forecast, and it is suggested that the Fourier transform mass spectrometer can meet the challenge. Success awaits a better understanding of the dynamics of high-mass ions. One route to improved understanding is outlined.

Gas-Phase Nazarov Cyclization of Protonated 2-Methoxy and 2-Hydroxychalcone: An Example of Intramolecular Proton-Transport Catalysis

Upon CA, ESI generated [M + H]+ ions of chalcone (benzalacetophenone) and 3-phenyl-indanone both undergo losses of H2O, CO, and the elements of benzene. CA of the [M + H]+ ions of 2-methoxy and 2-hydroxychalcone, however, prompts instead a dominant loss of ketene. In addition, CA of the [M + H]+ ions of 2-methoxy-β-methylchalcone produces an analogous loss of methylketene instead. Furthermore, the [M + D]+ ion of 2-methoxychalcone upon CA eliminates only unlabeled ketene, and the resultant product, the [M + D − ketene]+ ion, yields only the benzyl-d1 cation upon CA. We propose that the 2-methoxy and 2-hydroxy (ortho) substituents facilitate a Nazarov cyclization to the corresponding protonated 3-aryl-indanones by mediating a critical proton transfer. The resultant protonated indanones then undergo a second proton transport catalysis facilitated by the same ortho substituents producing intermediates that eliminate ketene to yield 2-methoxy- or 2-hydroxyphenyl-phenyl-methylcarbocations, respectively. The basicity of the ortho substituent is important; for example, replacement of the ortho function with a chloro substituent does not provide an efficient catalyst for the proton transports. The Nazarov cyclization must compete with an alternate cyclization, driven by the protonated carbonyl group of the chalcone that results in losses of H2O and CO. The assisted proton transfer mediated by the ortho substituent shifts the competition in favor of the Nazarov cyclization. The proposed mechanisms for cyclization and fragmentation are supported by high-mass resolving power data, tandem mass spectra, deuterium labeling, and molecular orbital calculations.

THE MECHANISM AND THERMODYNAMICS OF TRANSESTERIFICATION OF ACETATE-ESTER ENOLATES IN THE GAS PHASE

Abstract In solution, base-catalyzed hydrolysis and transesterification of esters are initiated by hydroxide- or alkoxide-ion attack at the carbonyl carbon. At low pressures in the gas phase, however, transesterification proceeds by an attack of the enolate anion of an acetate ester on an alcohol. Fourier transform mass spectrometry (FTMS) indicates that the reaction is the second-order process: − CH 2 -CO 2 -R + R′-OH → − CH 2 -CO 2 -R′ + R-OH and there is little to no detectable production of either alkoxide anion. Labeling studies show that the product and reactant enolate anion esters undergo exchange of hydrogens located α to the carbonyl carbon with the deuterium of R′-OD. The extent of the H/D exchange increases with reaction time, pointing to a short-lived intermediate. The alcoholysis reaction rate constants increase with increasing acidity of the primary, straight-chained alkyl alcohols, whereas steric effects associated with branched alcohols cause the rate constants to decrease. Equilibrium constants, which were determined directly from measurements at equilibrium and which were calculated from the forward and reverse rate constants, are near unity and show internal consistency. In the absence of steric effects, the larger enolate is always the favored product at equilibrium. The intermediate for the transesterification reaction, which can be generated at a few tenths of a torr in a tandem mass spectrometer, is tetrahedral, but other adducts that are collisionally stabilized under these conditions are principally loosely bound complexes.

Structural Determinant of Chemical Reactivity and Potential Health Effects of Quinones from Natural Products

Although many phenols and catechols found as polyphenol natural products are antioxidants and have putative disease-preventive properties, others have deleterious health effects. One possible route to toxicity is the bioactivation of the phenolic function to quinones that are electrophilic, redox-agents capable of modifying DNA and proteins. The structure-property relationships of biologically important quinones and their precursors may help understand the balance between their health benefits and risks. We describe a mass-spectrometry-based study of four quinones produced by oxidizing flavanones and flavones. Those with a C2-C3 double bond on ring C of the flavonoid stabilize by delocalization of an incipient positive charge from protonation and render the protonated quinone particularly susceptible to nucleophilic attack. We hypothesize that the absence of this double bond is one specific structural determinant that is responsible for the ability of quinones to modify biological macromolecules. Those quinones containing a C2-C3 single bond have relatively higher aqueous stability and longer half-lives than those with a double bond at the same position; the latter have short half-lives at or below ∼1 s. Quinones with a C2-C3 double bond show little ability to depurinate DNA because they are rapidly hydrated to unreactive species. Molecular-orbital calculations support that quinone hydration by a highly structure-dependent mechanism accounts for their chemical properties. The evidence taken together support a hypothesis that those flavonoids and related natural products that undergo oxidation to quinones and are then rapidly hydrated are unlikely to damage important biological macromolecules.