Evaluation of automated nano-electrospray mass spectrometry in the determination of non-covalent protein-ligand complexes

The use of electrospray ionization mass spectrometry (ESI-MS) for studying non-covalent interactions between macromolecules and ligands is well established. ESI-MS can be a useful tool for the determination of dissociation constants between molecules in the gas phase. We validate this method by studying the binding of the catalytic domain of cellobiohydrolase I (CBH I) from Trichoderma reesei to the disaccharide inhibitor cellobiose. The method was further applied to study two newly synthesized cellobiose derivatives (m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside and p-benzyloxybenzyl beta-cellobioside). In a titration experiment, peak areas of different charge states of the free enzyme and the complex were summed in order to determine the dissociation constant. For cellobiose and m-iodobenzyl 2-deoxy-2-azido-beta-cellobioside, the calculated values are in good agreement with those reported from either displacement titration or equilibrium binding experiments in solution. Due to non-specific binding, the dissociation constant of p-benzyloxybenzyl beta-cellobioside does not correspond with the solution-based value. Our results indicate the need for careful interpretation of data sets when using nanoESI to study non-covalent interactions.     

Chlorine--benzene complexes--the reliability of density functionals for non-covalent radical complexes

The structure of the chlorine atom-benzene complex has been a topic of significant controversy for more than 50 years. We have reexamined the structure of this complex with new density functional methods especially designed for non-covalent complexes, and compared the structures and energetics to those obtained using standard DFT and high accuracy composite methods. We find that the popular B3LYP functional fails to identify stationary points revealed by other functionals, and that the eta(1)-sigma complex appears to be more stable than the eta(1)-pi complex, contrary to other recent work, highlighting the careful selection of methods required in non-covalent radical systems.     

Fourier transform-ion cyclotron resonance mass spectrometric resolution,identification, and screening of non-covalent complexes of Hck Src homology 2 domain receptor and ligands from a 324-member peptide combinatorial library

The preferred ligands for the Hck Src homology 2 domain among a combinatorial library containing 324 different peptides were determined in a single experiment involving Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), electrospray ionization (ESI), stored-waveform inverse Fourier transformation (SWIFT), and infrared multiphoton laser disassociation (IRMPD). These were compared with the results obtained by conventional screening of the peptide library in solution using affinity chromatography. The results reported here show that by combining ESI, FT-ICR MS, SWIFT, and IRMPD, ligands likely to bind under physiological conditions are rapidly and efficiently identified, even from complex library mixtures. In the gas phase some discrimination against hydrophobic ligands could be observed. However, the illustrated feasibility of identifying high affinity ligand via gas-phase screening of complex library mixtures should lead to broad applications in the development of ligands for proteins with interesting biological activity, the first step that must be taken to develop a therapeutic agent.     

Membrane fouling in a constant-flux ultrafiltration cell

Factors affecting protein fouling of ultrafiltration membranes were investigated both in the presence and absence of a net transmembrane flux. The results obtained for the zero flux system showed that membrane-associated protein levels followed a saturation type isotherm having a maximum value of 207μg-cm⁻². In the presence of flux, membrane-associated protein showed an increase with increasing transmembrane flow. Under the conditions studied the membrane-associated protein eventually reached a flux-independent concentration which was a function of the tangential recycle flow. In all cases the limiting protein level was higher than the maximum level obtained in the absence of flux. The flow resistance of the fouling layer continued to increase after the membrane-associated protein had reached a limiting value, supporting the concept of a limited flux approach to modelling. The protein “adsorption” observed was considerable higher than could be explained by monolayer formation, and unlike Langmuir adsorption was shown to be freely reversible with changes in the hydrodynamic conditions.     

Evaluation of a pulsed EIMAC source for atomic-fluorescence spectrometry

An EIMAC xenon arc continuum source operated in the pulsed mode has been evaluated for potential use as a source of excitation in atomic-fluorescence spectrometry. The results were compared with those obtained with a similar lamp operated in the continuous-wave mode; little or no improvement was evident from this comparison. The lamp was found to be unstable in the pulsed mode, requiring the use of a rather large residual d.c. current in conjunction with a relatively low amplitude power-pulse in order to sustain the arc. Moreover, the pulsed source yielded a large background signal because of scattering of source-radiation in the flame; the background spectrum (with source "on") contained numerous large peaks due to fluorescence of molecular species in the flame and to irregularities in the spectral distribution of the pulsed lamp output transmitted to the signal detector by scatter.     

Electrospray ionisation mass spectrometric detection of weak non-covalent interactions in nogalamycin-DNA complexes

We demonstrate the use of electrospray ionisation mass spectrometry (ESI-MS) in high salt solutions for the analysis of weak non-covalent complexes of the anthracycline antibiotic nogalamycin with novel DNA hairpin structures; high signal-to-noise ratios for the complexes in the absence of bound Na+ ions permits relative binding affinities to be estimated.     

Multidentate ligands for the synthesis of multi-metallic complexes

The reaction of the Schiff base species tris-((2-hydroxybenzylidene)aminoethyl)-amine (TrenSal) and tris-((2-hydroxy-5-bromobenzylidene)aminoethyl)amine (Tren5BrSal) with the acetates of nickel and zinc are reported. Two trimetallic complexes (M₃L₂) of Tren5BrSal with nickel and zinc have been crystallographically characterised. The attempted crystallisation of bis-(tris-((2-hydroxybenzylidene)aminoethyl)amine nickel) nickel from solutions containing TMEDA lead to the production of two novel complexes: namely a nickel adduct of the partially hydrolysed TrenSal ligand and an interesting nickel bromide–carbonate salt. [(TrenSal)₂Ni₃] is reacted with PbCl₂ to form a novel tetrametallic complex, [{(TrenSal)Ni}Pb(NC₅H₅)Cl]₂, where a Pb₂Cl₂ moiety replaces the nickel at the core of the complex. Extending the study to include the related hexadentate ligand, 1,1,1-tris-((2-hydroxybenzylidene)-aminomethyl)propane (TEtSal), we were able to isolate and characterise both [(TEtSal)₂Ni₃] and [{(TEtSal)Ni}₂Pb].     

Characterization of a micro spiral flow cell for chemiluminescence detection

A 5.5 μl spiral micro-flow cell, mounted in front of a photomultiplier, is made from Teflon capillary (75 cm×100 μm ID) with two inlets for the CL reagent and carrier buffer and a waste outlet. It allows the rapid mixing of CL reagent and analyte and simultaneous detection of the emitted light. Using a flow rate of 25 μl/min for a 0.4 mM luminol-8 μM hemin solution (pH 11.6) and 50 μl/min of carrier buffer (pH 11.6), the slight exponential calibration curve for the flow injection–chemiluminescence (FI–CL) determination of H₂O₂ is 2.5–10 μM and the detection limit is 1.5 μM. The detection limit achieved by using a spiral flow cell is 24 times lower than that obtained from a conventional FI system with a low dead volume tee mixer and a 12 μl flow cell in a HPLC fluorometer with the source lamp off. This luminol CL detection method is successfully applied to the enzymatic determination of -lactate by FI. The lactate sample is mixed with polyethylene glycol (PEG)-stabilized lactate oxidase (LO) enzyme and then injected into the buffered (pH 7.5) carrier stream for CL detection of the H₂O₂ product. Using the optimal conditions of reaction temperature set to 37.5 °C and flow rates of 45 μl/min for the CL reagent and 60 μl/min for the carrier buffer, the calibration range for lactate is 5–50 μM and the detection limit is 2.9 μM. This method is applied to the determination of -lactate in beer.     

Evaluation of a pulsed flash-tube for inductively-coupled plasma atomic-fluorescence spectrometry

A pulsed continuum xenon flash-tube is used as an excitation source in ICP-AFS. The simultaneous excitation of many elements is attractive and avoids the one source per element as in conventional ICP-AFS. In this study, the pulsed continuum flash-tube is evaluated as an excitation source for multielement atomic fluorescence spectrometry in an ICP. Analytical figures of merit are given for the elements studied.     

The use of ultrafiltration for the separation and fractionation of organic ligands in fresh waters

The effects of various experimental conditions on the results obtained by using membrane filtration for the separation and fractionation of organic matter in fresh waters (mainly fulvic and humic substances) are described. The technique used (washing or concentration) and the initial concentration of the organic matter to be filtered are the most critical factors. The technique is used for the fractionation of eight water samples, one sample of peat interstitial water, five water extracts of soil and four water samples obtained by decomposition of leaves. The results are compared. A comparison is also made with results cited in the literature.     

Using non-covalent complexes to direct the fragmentation of glycosidic bonds in the gas phase

An investigation of the gas phase chemistry of proton bound oligosaccharide (S)-ligand (L) non-covalent complexes, [S + H + L](+) has been carried out using electrospray ionization (ESI) and tandem mass spectrometry in a quadrupole ion trap. When subjected to collision-induced dissociation (CID), these [S + H + L](+) complexes undergo a range of reactions that can be broadly classified into three main types: (1) Simple dissociation into the individual monomers; (2) cleavage of the oligosaccharide to form B-type sequence ions; (3) cleavage of the ligand species. The second type of reaction is particularly interesting as it can produce a "ladder series" of [B(x) + L](+) ions via ligand induced oligosaccharide bond cleavage. This novel gas phase reaction greatly simplifies the sequencing of oligosaccharides. Both the oligosaccharide and ligand were found to influence the type of reaction pathway observed, with the "ladder series" of [B(x) + L](+) ions being favored for permethylated oligosaccharides and for bifunctional ligands. Cytosine is a particularly good ligand at facilitating the formation of [B(x) + L](+) ions. Analogies with condensed phase chemistry of sugars is made and a potential mechanism for ligand induced oligosaccharide bond cleavage is proposed.     

Optimisation of the selectivity of a pulsed flame photometric detector for unknown compound screening

Identification of traces of chemical warfare agents (CWA) is generally performed with spectral methods such as mass spectrometry (MS) or NMR, but the use of element-specific detectors is most often required to extract interesting signals from gas chromatographic (GC) data heavily masked by natural interferents. The pulsed flame phosphorus detector (P-FPD) is able to detect phosphorus, sulphur and arsenic, and thus should be very well-suited to CWA detection. However, first results using standard operating conditions recommended by the manufacturer of P-FPD led to false positive detection of phosphorus-containing compounds on the sulphur line. Therefore, an optimisation process of the selectivity of P-FPD for phosphorus versus sulphur or arsenic was undertaken, and allowed to identify gate delay and gate width as crucial parameters for the performance of P-FPD. While selectivity could be significantly improved, unexpectedly, this resulted in a concurrent important loss of sensitivity (ca. 45%) for arsenic, which suggests that this detector should be carefully optimised with respect, and prior to its purported use.     

Reactions of coordinated ligands in uranyl hydroxylaminate complexes

The reactions of coordinated ligands in uranyl complexes were studied for the first time. The reaction of uranyl hydroxylaminate complexes with aldehyde and ketones of different structure was shown to result in the formation of corresponding oxime complexes.     

Introduction of new ancillary ligands to the iridium complexes having 2,3-diphenylquinolinato ligands for OLED

We investigated the effect of an ancillary ligand (AL) on the emission color and luminous efficiencies of its complex, Ir(4-Me-2,3-dpq)₂(AL), where 4-Me-2,3-dpq represents 4-methyl-2,3-diphenylquinolinato ligand. We expected that ancillary ligand modification by introduction of the bulky substituent to the complexes might allow luminous efficiency increase by reduction of T–T annihilation. Furthermore, some ancillary ligands may contribute to fine-tuning of their complex emission colors by influencing the energy level of Ir d-orbitals upon the orbital mixing. As new ancillary ligands substituting for acac which is a typical AL in the iridium complexes, pyrazolone-based ligands, 4-R-5-methyl-2-phenyl-2,4-dihydro-pyrazol-3-one series (przl-R), were prepared, where R represents C₆H₅, C₆H₄CH₃ and C₆H₄Cl. These ligands were chelated to the iridium center to yield a new series of the iridium complexes, Ir(4-Me-2,3-dpq)₂(przl-R). The X-ray crystal structure of Ir(4-Me-2,3-dpq)₂(przl-C₆H₄Cl) was determined. The electrochemical and luminescence properties of the iridium complexes were investigated. The effect of the przl-substituents on the emission colors of the complexes was not significant. On the other hand, the luminous efficiencies of Ir(4-Me-2,3-dpq)₂(przl-C₆H₅) and Ir(4-Me-2,3-dpq)₂(przl-C₆H₄CH₃) were higher than that of Ir(4-Me-2,3-dpq)₂(acac).     

Virtual screening for ligands of the insect molting hormone receptor

Insect growth is regulated by the orchestrated event of ecdysteroids and their receptor proteins. Agonists/antagonists of ecdysteroid receptor are predicted to disrupt normal growth, providing good candidates of new insecticides. A database of over 2 million compounds was subjected to a shape-based virtual screening cascade to identify novel nonsteroidal hits similar to the known EcR ligand ponasterone A. Testing revealed micromolar hits against two strains of insect cells. Docking experiments against EcR were used to support the predicted binding mode of these ligands based on their overlay to ponasterone A.