Preservation of non-covalent associations in electrospray ionization mass spectrometry: Multiply charged polypeptide and protein dimers

The ‘softness’ of the electrospray ionization (ESI) method provides a direct link between solution chemistry and the inherent gas-phase environment of mass Spectrometry. Available results related to the preservation of non-covalent associations into the gas phase after ESI are reviewed. These associations include the possible retention of elements of higher order protein structure, non-covalent polypeptide–heme associations and enzyme complexes. Experimental results are presented showing that non-covalently bound polypeptide and protein dimer ions are relatively common as low level contributions to ESI mass spectra. It is argued that these dimers are reflective of multimeric species in solution since Coulombic barriers preclude dimerization after ESI although uncertainty remains regarding whether they exist prior to the formation of highly charged droplets. The dissociation of dimers is facile and for proteins can yield monomers having a broad distribution of charge states. The detection of non-covalently associated dimers requires gentle ESI mass spectrometer interface conditions, yielding relatively low levels of internal excitation. Under such conditions incomplete molecular ion desolvation can result in experimental artifacts for tandem mass spectrometric experiments. ESI mass Spectrometry may have broad potential for the study of noncovalent liquid phase associations.     

On the liquid chromatography and identification of the flavonoids,present in the “sumach tannic acid” extracted fromRhus coriaria

Summary The leaves ofRhus coriaria contain about 15–20% polyphenolic compounds. These are mostly hydrolysable tannins, with a central glucose unit, to which several gallic acid rests are bound depsidically. 5 to 10% of the total polyphenolic fraction however, consists of condensed tannins or flavonoids. This work studies the identity and the liquid chromatographic behaviour of these flavonoids. The presence of the dimeric flavonoids agathisflavone, amenthoflavone and hinokiflavone is proved. A new dimeric flavanoid (Sumaflavone) is tentatively identified.     

Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling

The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.     

Virtual two-dimensional gel electrophoresis of high-density lipoproteins

High-density lipoproteins (HDLs) isolated by immunoaffinity chromatography and separated by immobilized pH gradient-isoelectric focusing (IPG-IEF) were examined by mass spectrometry directly, applying a new proteomics technology, virtual two-dimensional (2-D) gel electrophoresis. A preliminary examination of HDL particles has revealed at least 42 unique masses for protein species with isoelectric points between pH 5.47-5.04, some of which have not been observed previously. By delivering masses of intact proteins from complex cellular mixtures in a format that correlates directly to classical 2-D gel analyses, virtual 2-D gel electrophoresis constitutes a general discovery tool to expose and monitor protein isoforms and post-translational modifications. Furthermore, its general ability to deliver ions from sub-picomole level proteins enmeshed in complex cellular mixtures potentially fulfills the need of top-down proteomics to obtain intact protein ions from microscale samples. Additional comparison of such data to 2-D gel analyses and their identified proteins may elucidate the functions of the individual apolipoprotein components and the cardioprotective effects of HDL.     

Determination of sulfur components in natural gas: A review

More stringent environmental regulations as well as higher demands presently being imposed on the sulfur content of natural gas feed-stocks for chemical processes necessitate the development of new analytical procedures for sulfur determination in natural gas. Only analytical procedures based on gas chromatography can meet the sensitivity and accuracy requirements dictated by environmental regulation institutions and modern chemical industry. The complexity of the natural gas matrix as well as the extremely low concentration levels at which the sulfur species occur make the development of these analytical methods a true challenge. In this review the three steps common for analytical methods for trace analysis in complex matrices, i.e. pretreatment, chromatographic separation, and detection, are discussed in detail. Possible methods for calibration of the system are discussed in the final section. Various techniques to determine sulfur in natural gas are described. Depending on the application, the most suitable system has to be selected. For example, for on-line application in a hazardous area a simple and rugged system is required, i.e. a simple gas chromatograph with a flame photometric detector, while for laboratory application a more complex instrument including preconcentration, column switching, and more exotic detection systems could be more suitable. Therefore it is crucial to define the requirements of the instrument at an early stage and use the information in this review article to develop/select a dedicated instrument/procedure for the problem at hand.     

Morphology and mechanical properties of a novel amorphous polyamide/nanoclay nanocomposite

A novel amorphous polyamide/montmorillonite nanocomposite based on poly(hexamethylene isophthalamide) was successfully prepared by melt intercalation. Wide angle X-ray diffraction and transmission electron microscopy showed that organoclay containing quaternary amine surfactants with phenyl and hydroxyl groups was delaminated in the polymer matrix resulting in well-exfoliated morphologies even at high montmorillonite content. Differential scanning calorimetry results indicated that clay platelets did not induce the formation of a crystalline phase in this amorphous polymer. Tensile tests demonstrated that the addition of nanoclay caused a dramatic increase in Young's modulus (almost twofold) and yield strength of the nanocomposites compared with the homopolymer. The nanocomposites exhibited ductile behavior up to 5 wt % of nanoclay. The improvement in Young's modulus is comparable with semicrystalline aliphatic nylon 6 nanocomposites. Both the main chain amide groups and the amorphous nature of the polyamide are responsible for enhancing the dispersion of the nanofillers, thereby, leading to improved properties of the nanocomposites. The structure-property relationship for these nanocomposites was also explored. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 2605–2617, 2008     

Anionic cross-coupling reaction of alpha-metallated alkenyl sulfoximines and alkenyl sulfoximines with cuprates featuring a 1,2-metal-ate rearrangement of sulfoximine-substituted higher order alkenyl cuprates and an alpha-metallation of alkenyl sulfoximines by cuprates

(E)- and (Z)-configured alpha-lithioalkenyl sulfoximines, which are available through lithiation of the corresponding alkenyl sulfoximines, undergo a anionic cross-coupling reaction (ACCR) with organocuprates with formation of the corresponding alkenyl cuprates and sulfinamide. The alkenyl cuprates can be trapped by electrophiles. The ACCR presumably proceeds via the formation of a higher-order sulfoximine-substituted alkenyl cuprate, which undergoes a 1,2-metal-ate rearrangement whereby the sulfoximine group acts as the nucleofuge. The parent (E)- and (Z)-configured alkenyl sulfoximines suffer upon treatment with an organocuprate a deprotonation at the alpha-position with formation of the corresponding alpha-cuprioalkenyl sulfoximines. These derivatives also enter into a similar ACCR with organocuprates. The ACCR of sulfoximines substituted homoallylic alcohols allows a stereoselective access to enantio- and diastereopure substituted homoallylic alcohols.     

Probing occupied states of the molecular layer in Au-alkanedithiol-GaAs diodes

Internal photoemission (IPE) studies were performed on molecular diodes in which the alkanedithiol [HS(CH(2))(n)SH, n = 8, 10] molecular layer is sandwiched between Au and GaAs electrodes. The results are compared to those from Au-GaAs Schottky diodes. An exponential energy dependence in the IPE yield was observed for the molecular diodes, in contrast to the quadratic energy dependence characteristic of metal-semiconductor Schottky diodes, indicating that Au is not the source of electrons in the IPE process in the molecular diodes. From the GaAs dopant density dependence, we also can rule out GaAs being the source of these electrons. Compared with the results of cluster electronic structure calculations, we suggest that IPE is probing the occupied levels of GaAs-molecular interfacial states.     

Direct measurement of the radiative lifetime of vibrationally excited OH radicals

Neutral molecules, isolated in the gas phase, can be prepared in a long-lived excited state and stored in a trap. The long observation time afforded by the trap can then be exploited to measure the radiative lifetime of this state by monitoring the temporal decay of the population in the trap. This method is demonstrated here and used to benchmark the Einstein A coefficients in the Meinel system of OH. A pulsed beam of vibrationally excited OH radicals is Stark decelerated and loaded into an electrostatic quadrupole trap. The radiative lifetime of the upper Lamda-doublet component of the Chi2Pi3/2, v=1, J=3/2 level is determined as 59.0+/-2.0 ms, in good agreement with the calculated value of 58.0+/-1.0 ms.