bluuebunny/crossref_metadata_2025 huggingface dataset:
A proteomic analysis of 14‐3‐3 binding proteins from developing barley grains
Authors: Alexander Ross D. , Morris Peter C.
Published: February 2006

14‐3‐3 proteins are important eukaryotic regulatory proteins. Barley (Hordeum vulgare L.) 14‐3‐3A was over‐expressed, immobilised and used to affinity purify 14‐3‐3 binding proteins from developing barley grains. Binding was shown to be phosphorylation‐dependent. These proteins were fractionated by PAGE and identified by MALDI‐TOF MS. In total, 54 14‐3‐3 binding proteins were identified, 49 of these interactions are novel to plants. These proteins fell into a number of functional categories. The largest category was for carbohydrate metabolism, including plastidic enzymes for starch synthesis and modification. 14‐3‐3 was shown to be present in isolated plastids. Four of five enzymes involved in sucrose biosynthesis from triose phosphates were identified, suggesting co‐ordinated regulation of this pathway. Invertase and sucrose synthase, which break down sucrose to hexoses, were found. Sucrose synthase activity was shown to be inhibited by exogenous 14‐3‐3 in a dosage‐dependent manner. The second‐largest functional group was for proteins involved in stress and defence responses; for example, RGH2A, closely related to the MLA powdery mildew resistance protein, was found. This work illustrates the broad range of processes in which 14‐3‐3 may be involved, and augments previous data demonstrating key roles in carbohydrate metabolism and plant defence.
Protein interaction networks of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster: Large‐scale organization and robustness
Authors: Li Dong , Li Jianqi , Ouyang Shuguang , Wang Jian , Wu Songfeng , Wan Ping , Zhu Yunping , Xu Xiaojie , He Fuchu
Published: February 2006

No abstracts.
Acrylic polymer/silica hybrid for electron‐beam resists
Authors: Tamai Toshiyuki , Matsuura Yukihito , Watanabe Mitsuru , Matsukawa Kimihiro
Published: February 2006

An acrylic polymer/silica hybrid resist film was investigated for fabricating a microstructure by electron‐beam (EB) lithography. EB lithography on the hybrid thin film afforded a positive pattern whose depth corresponded to the EB exposure dose; this indicated that the hybrid was an analog resist and could fabricate a three‐dimensional microstructure. The resist film had high heat resistance and compatibility with the underlying quartz plate, probably because of the silica component. The acrylic polymer/(RSiO1.5)n hybrid film showed higher EB sensitivity than a film of the crosslinked acrylic polymer and an acrylic polymer/(SiO2)n hybrid. Atomic force microscopy observation of the hybrid film surface showed the homogeneous dispersion of the acrylic polymer and the silica components in the hybrid film. The acrylic polymer component was EB‐sensitive, whereas dispersing the acrylic polymer and silica components homogeneously also played an important role in increasing the EB sensitivity. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2107–2116, 2006
Synthesis and characterization of macrocyclic vinyl aromatic polymers
Authors: Hogen‐Esch Thieo E.
Published: February 2006

The synthesis and characterization by size exclusion chromatography, liquid chromatography, NMR, matrix‐assisted laser desorption/ionization, thermal analysis, and other techniques of well‐defined and narrow molecular weight distribution macrocyclic polystyrene (PS), poly(2‐vinylpyridine), poly(α‐methylstyrene), poly (2‐vinyl‐naphthalene) (P2VN), and poly(9,9‐dimethyl‐2‐vinylfluorene) (PDMVF) containing a single 1,4‐benzylidene, methylidene, or 9,10‐anthracenylidene unit are reviewed. The absorption and emission spectroscopy of PS, P2VN, and PDMVF is also discussed. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 2139–2155, 2006
Demographic and clinical predictors of preparatory grief in a sample of advanced cancer patients
Authors: Mystakidou Kyriaki , Tsilika Eleni , Parpa Efi , Katsouda Emmanuela , Sakkas Palvos , Galanos Antonis , Vlahos Lambros
Published: February 2006

Background: Preparatory grief encompasses grief for losses that have already occurred, are currently being experienced, and losses that will or might ensue in the future after the death, as a consequence of it.

Aim: To examine the relative contribution of demographic and clinical variables in predicting cancer patients' preparatory grief as recorded from the Preparatory Grief in Advanced Cancer Patients (PGAC) scale. Moreover, researchers were interested in determining whether these dimensions were independently and uniquely associated with preparatory grief.

Methods: Two hundred advanced cancer patients treated in a Pain Relief and Palliative Care Unit completed the PGAC scale, while researchers recorded data on demographic characteristics, disease status and treatment regimen.

Results: The analyses showed that the most significant correlations were found between preparatory grief and age (r=−0.227, p=0.001), gender (p=0.006), family status (p=0.019), performance status (p=0.010), surgery (p=0.029), opioids (p=0.019), and diagnosis (p=0.038). In the prediction of preparatory grief, the contribution of age, performance status, history of other surgery, gender and opioids is high.

Conclusions: Awareness of the specific patients' demographic and medical characteristics, such as old age, poor performance status, history of other surgery, female gender, and strong opioids, contribute to the prediction of patients' preparatory grief. Copyright © 2006 John Wiley & Sons, Ltd.
Molecular modeling and inhibitory activity of cowpea cystatin against bean bruchid pests
Authors: Aguiar Juliana M. , Franco Octávio L. , Rigden Daniel J. , Bloch Carlos , Monteiro Ana C.S. , Flores Victor M.Q. , Jacinto Tânia , Xavier‐Filho José , Oliveira Antonia E.A. , Grossi‐de‐Sá Maria F. , Fernandes Kátia V.S.
Published: February 2006

Plant cystatins show great potential as tools to genetically engineer resistance of crop plants against pests. Two important potential targets are the bean weevilsAcanthoscelides obtectusandZabrotes subfasciatus, which display major activities of digestive cysteine proteinases in midguts. In this study a cowpea cystatin, a cysteine proteinase inhibitor found in cowpea (Vigna unguiculata) seeds, was expressed inEscherichia coliand purified with a Ni‐NTA agarose column. It strongly inhibited papain and proteinases from midguts of bothA. obtectusandZ. subfasciatusbruchids, as seen by in vitro assays. When the protein was incorporated into artificial seeds at concentrations as low as 0.025%, and seeds were consumed by the bruchids larva, dramatic reductions in larval weight, and increases in insect mortality were observed. Molecular modeling studies of cowpea cystatin in complex with papain revealed that five N‐terminal residues responsible for a large proportion of the hydrophobic interactions involved in the stabilization of the enzyme–inhibitor complex are absent in the partial N‐terminal amino acid sequencing of soybean cystatin. We suggest that this structural difference could be the reason for the much higher effectiveness of cowpea cystatin when compared to that previously tested phytocystatin. The application of this knowledge in plant protein mutation programs aiming at enhancement of plant defenses to pests is discussed. Proteins 2006. © 2006 Wiley‐Liss, Inc.
The fragment transformation method to detect the protein structural motifs
Authors: Lu Chih‐Hao , Lin Yeong‐Shin , Chen Yu‐Ching , Yu Chin‐Sheng , Chang Shi‐Yu , Hwang Jenn‐Kang
Published: February 2006

To identify functional structural motifs from protein structures of unknown function becomes increasingly important in recent years due to the progress of the structural genomics initiatives. Although certain structural patterns such as the Asp‐His‐Ser catalytic triad are easy to detect because of their conserved residues and stringently constrained geometry, it is usually more challenging to detect a general structural motifs like, for example, the ββα‐metal binding motif, which has a much more variable conformation and sequence. At present, the identification of these motifs usually relies on manual procedures based on different structure and sequence analysis tools. In this study, we develop a structural alignment algorithm combining both structural and sequence information to identify the local structure motifs. We applied our method to the following examples: the ββα‐metal binding motif and the treble clef motif. The ββα‐metal binding motif plays an important role in nonspecific DNA interactions and cleavage in host defense and apoptosis. The treble clef motif is a zinc‐binding motif adaptable to diverse functions such as the binding of nucleic acid and hydrolysis of phosphodiester bonds. Our results are encouraging, indicating that we can effectively identify these structural motifs in an automatic fashion. Our method may provide a useful means for automatic functional annotation through detecting structural motifs associated with particular functions. Proteins 2006. © 2006 Wiley‐Liss, Inc.
Terminal ion pairs stabilize the second β‐hairpin of the B1 domain of protein G
Authors: Huyghues‐Despointes Beatrice M. P. , Qu Xiaotoa , Tsai Jerry , Scholtz J. Martin
Published: February 2006

The effects of terminal ion pairs on the stability of a β‐hairpin peptide corresponding to the C‐terminal residues of the B1 domain of protein G were determined using thermal unfolding as monitored by nuclear magnetic resonance and circular dichroism spectroscopy. Molecular dynamics (MD) simulations were also performed to examine the effect of ion pairs on the structures. Eight peptides were studied including the wild type (G41) and the N‐terminal modified sequences that had the first residue deleted (E42), replaced with a Lys (K41), or extended by an additional Gly (G40). Acetylated variants were made to examine the effect of removing the positive N‐terminal charge on β‐hairpin stability. The rank in stability determined experimentally is K41 > E42 ≈ G41 ≈ G40 > Ac‐K41 > Ac‐E42 ≈ Ac‐G41 > Ac‐G40. The Tm of the K41 peptide is 12 °C higher than G41, while the Tm values for the acetylated peptides are less than their unacetylated forms by more than 15 °C. NOE cross‐peaks between side‐chain methylene groups at the N‐ and C‐termini and larger CαH shifts compared to random values are seen for K41. The addition of 20% methanol increases the stability in K41 and G41. The MD studies complement these results by showing that the charged N‐terminus is important to stability. The type of ion pair observed varies with peptide, and when formed the simulations show that the ion pair can prevent fraying of the β‐strands through electrostatic and hydrophobic contacts. Therefore, introducing favorable electrostatic interactions at the N‐ and C‐termini can substantially enhance β‐hairpin stability and help define the structure. Proteins 2006. © 2006 Wiley‐Liss, Inc.
Investigating the in situ degradation of atrazine in groundwater
Authors: Pearson Robert , Godley Andrew , Cartmell Elise
Published: February 2006

This study focused on whether or not atrazine could be degraded by indigenous groundwater bacteria as part of an in situ remediation approach. Groundwater was taken from an unconfined middle upper chalk site where concentrations of atrazine and nitrate were typically in the ranges 0.02–0.2 µg litre−1 and 11.6–25.1 mg NO3‐N litre−1 respectively. Sacrificial batch studies were performed using this groundwater spiked with atrazine at a concentration of 10 µg litre−1 in conjunction with a minimal mineral salts liquid (Glu‐MMSL) medium which contained glucose as the sole carbon source. Treatments comprised either the Glu‐MMSL groundwater cultured bacteria or Pseudomonas sp. strain ADP. Results from sacrificial batches indicated the occurrence of bacterial growth and denitrification as monitored by optical density (absorbance at 600 nm) and NO3‐N content. Analysis of atrazine content by solid phase extraction coupled with high‐performance liquid chromatography showed no degradation of atrazine over a period of 103 days in either treatment. These results indicated that no acclimatised bacterial community featuring positive degraders to the herbicide atrazine had become established within this chalk aquifer in response to the trace levels encountered. Copyright © 2006 Society of Chemical Industry
The effect of insecticide application sequences on the control and insecticide resistance status of the peach‐potato aphid, Myzus persicae (Hemiptera:Aphididae), on field crops of potato
Authors: Parker William E , Howard Julia J , Foster Stephen P , Denholm Ian
Published: February 2006

Experiments were done on commercial potato crops in the UK to investigate the effect of different insecticide sequences on the control and insecticide resistance status of Myzus persicae (Sulzer). The work was done to provide field validation of similar laboratory studies done in ‘field simulators’. To ensure adequate aphid populations and to influence the initial resistance status of the aphid population, cultured M. persicae from a clone of known resistance status (esterase R1, kdr heterozygote, non‐MACE (modified acetylcholinesterase)) were inoculated into both experiments. Two‐spray programmes starting with λ‐cyhalothrin (a pyrethroid insecticide) gave poor control in comparison with programmes starting with pirimicarb (a carbamate insecticide) or pirimicarb‐containing mixtures. This concurred closely with the results obtained from single applications in field simulator studies. Treatment sequences containing pymetrozine (a pyridine azomethine insecticide) were also effective, though slower‐acting. This again concurs with field simulator studies. The proportions of aphids carrying different resistance mechanisms were largely unaffected by treatment in these experiments. The implications of these results for field control strategies are discussed. Copyright © 2006 Society of Chemical Industry
Improved efficacy of ethyl formate against stored grain insects by combination with carbon dioxide in a ‘dynamic’ application
Authors: Haritos Victoria S , Damcevski Katherine A , Dojchinov Greg
Published: February 2006

Ethyl formate is being evaluated as a fumigant for stored grain as it is a potential alternative to the ozone‐depleting fumigant methyl bromide and to phosphine, which is under pressure owing to the development of strong resistance in stored grain insects. However, use of ethyl formate faces significant challenges, such as poor penetration through grain, significant losses to grain sorption, high concentrations of fumigant required to control insects, and flammability risks, which have limited its further development. In this study it was found that the combination of carbon dioxide (5–20%) with ethyl formate significantly enhanced efficacy of the fumigant against external living stages of the rice weevil, Sitophilus oryzae (L.), the lesser grain borer, Rhyzopertha dominica F., and the flour beetle, Tribolium castaneum (Herbst). Dynamic application of ethyl formate and carbon dioxide mixture (100 mg litre−1 ethyl formate, 20% CO2) pumped through a model silo containing wheat (50 kg) for one gas exchange was also investigated. A flow rate of 6 litres min−1 gave a relatively even distribution of fumigant throughout the grain column and similar mortality levels among cultures of S. oryzae and T. castaneum placed at three positions, the top, middle and bottom of the column. Mortality of 99.8% of mixed stage cultures of T. castaneum and 95.1% of S. oryzae was achieved in 3 h exposures to 111 and 185 mg ethyl formate h litre−1 respectively applied by the dynamic method. It is concluded that the combination of carbon dioxide with ethyl formate and dynamic application enhances distribution and efficacy of the fumigant against stored grain insects. Copyright © 2006 Society of Chemical Industry
Use of oils combined with low doses of insecticide for the control of Myzus persicae and PVY epidemics
Authors: Martín‐López Begoña , Varela Ianire , Marnotes Silvia , Cabaleiro Cristina
Published: February 2006

Experiments were carried out in the laboratory to assess the insecticidal effect on Myzus persicae Sulzer of different oils applied alone or combined with imidacloprid or pirimicarb. The oils tested were a horticultural mineral oil, a refined rapeseed oil, a refined soya oil and a raw fish oil. When the oils were sprayed alone on pepper plants infested with M. persicae, mineral oil caused the highest mortality of aphids (over 80%). Applied before aphid infestation of pepper leaves and in mixture with low doses of imidacloprid (at one‐fifth of the dose recommended by the manufacturer) and pirimicarb (at one‐tenth of the dose recommended by the manufacturer), the oils did not significantly increase the toxicity of the insecticides alone. However, sprayed on aphid‐infested pepper plants, the mortality rates achieved by imidacloprid/mineral oil and imidacloprid/rapeseed oil mixtures were significantly higher than those achieved by imidacloprid alone at 16 and 24 h. In a field experiment the effect on the incidence of the potato virus (PVY) of the oils in combination with imidacloprid was determined. Mineral oil, rapeseed oil and soya oil were sprayed eight times onto seed potato plants treated with imidacloprid before sowing. Mineral oil reduced PVY‐infected plants by 60% and rapeseed oil by 40% compared with plots treated with imidacloprid. The oils applied as 10 ml litre−1 emulsions in water did not cause symptoms of phytotoxicity on the potato plants, and yield was not reduced. Copyright © 2006 Society of Chemical Industry
Studies on the structure and conduction mechanism of (Acrd)[Au(mnt)2]2.7 and (Phenz)[Au(mnt)2]3 molecular single crystals
Authors: Hafiz Hafiz Rashad
Published: February 2006

Two quasi‐one‐dimensional acridinium (Acrd) and phenazinium (Phenz) molecular conductors with a single anion of maleonitriledithiolato aurate (Au(mnt)2) were obtained by electrochemical crystallization. The Acrd single crystal was found to have a non‐stoichiometric composition donor (D):acceptor (A) = 1:2.7, whereas the Phenz single crystal was found to have a stoichiometric composition D:A = 1:3. Crystal structure analysis revealed that the former has a monoclinic lattice with a = 9.26, b = 7.63, c = 27.76, β = 104.8, Z = 2, and the latter has a monoclinic lattice with a = 17.06, b = 14.46, c = 28.30, β = 108.3, Z = 6. The Acrd radicals were assumed to form alternate stacking with the anion sublattice due to the appearance of satellite reflections indicating a sinusoidal modulation structure. The Phenz single crystal was verified as adopting cation stacks with disorder in the anion sublattice. The results were supported by FTIR spectroscopy and cyclovoltammetry measurements. The room temperature dc conductivity of the Phenz crystal was found to be about two orders of magnitude higher than that of the Acrd crystal. A quasi‐one‐dimensional conduction mechanism was proposed for the Phenz crystal but a conventional nearest‐neighbour hopping mechanism was shown to best describe conduction in the Acrd single crystal exhibiting a well‐defined metal‐to‐insulator transition. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Resonant cavity enhanced optical microsensor for molecular interactions based on porous silicon
Authors: De Stefano Luca , Rea Ilaria , Rendina Ivo , Rotiroti Lucia , Rossi Mosè , D'Auria Sabato
Published: February 2006

The molecular binding between the glutamine binding‐protein (GlnBP) from Escherichia coli and L‐glutamine (Gln) is detected by means of an optical biosensor based on porous silicon technology. The binding event is optically transduced in the wavelength shift of the porous silicon optical microcavity (PSMC) reflectivity spectrum. The hydrophobic interaction links the GlnBP, which acts as a molecular probe for Gln, to the hydrogenated porous silicon surface area. We can thus avoid any preliminary surface functionalization process. The protein infiltrated PSMC results stable to oxidation at least for few cycles of wet measurements. The penetration of the proteins into the pores of the porous silicon matrix has been optimized: a strong base post‐etch process increases the pore size and removes any nanostructure on top and inside the porous silicon multilayer while does not degrade the optical response and the quality of the microcavity. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Effects of rapid thermal processing on oxide precipitation in conventional and nitrogen‐doped Czochralski silicon
Authors: Ma Xiangyang , Fu Liming , Tian Daxi , Cui Can , Yang Deren
Published: February 2006

In this paper, the effects of vacancies introduced by rapid thermal processing (RTP) on nucleation and growth of oxide precipitates in Czochralski (Cz) silicon are elucidated. Moreover, the nitrogen and vacancy enhancement roles in oxide precipitation are differentiated through designing specific annealing schemes. Furthermore, it is pointed out that the RTP‐based internal gettering process for a nitrogen‐doped Cz silicon wafer should be different from that for a conventional Cz silicon wafer in order to strengthen the intrinsic gettering capability. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Ge‐dots/Si multilayered structures fabricated by Ni‐induced lateral crystallization
Authors: Shi Y. , Yan B. , Pu L. , Zhang K. J. , Han P. , Zhang R. , Zheng Y. D.
Published: February 2006

We report on the fabrication of high‐quality Ge‐dots/Si multilayered structure films by metal‐induced lateral crystallization (MILC). Ge dots are self‐assembled grown on amorphous Si layer using low‐pressure chemical vapor deposition, and then low‐temperature crystallization is carried out by Ni‐based MILC processing. The micro Raman spectroscopy, optical microscopy and electron microscopy observations reveal that the crystallized Si film has large leaf‐like grains elongated along the MILC direction with (110) preference. Moreover, the strain shift of Ge dots reflects the formation of high quality interface between the crystallized Si and Ge dot. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Involvement of iron–phosphorus complexes in iron gettering for n‐type silicon
Authors: Mchedlidze T. , Kittler M.
Published: February 2006

Mechanisms for phosphorus (P) diffusion gettering (PDG) for iron are supplemented by possible formation of iron–phosphorus complexes in heavy P‐doped region. Existence of such complexes was recently reported based on the results of electron‐spin resonance investigations. DLTS measurements suggest a high probability for the formation of iron–phosphorus complexes in n‐type silicon in the presence of vacancies and/or vacancy–phosphorus pairs. On the other hand, recent theoretical calculations predict formation of negatively charged vacancy–phosphorus pairs in the heavily P‐doped region of silicon during PDG. These facts indicate on possibility to explain the high efficiency of the PDG process for iron by assuming formation of iron–phosphorus complexes in the heavy P‐doped region of silicon. Possible advantages of application of hydrogen or nitrogen assisted PDG are considered. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
A contribution to oxide precipitate nucleation in nitrogen doped silicon
Authors: Kissinger G. , Lambert U. , Weber M. , Bittersberger F. , Müller T. , Richter H. , von Ammon W.
Published: February 2006

Based on Fourier transform infrared (FTIR) spectroscopy and bulk micro‐defect investigations, in relation to earlier results of other groups, we suggest the following model for oxide precipitate nucleation in N‐doped silicon. Around 600 °C a nucleation maximum exists where oxide precipitates are formed via oxygen attachment to both NOO and NNO complexes. These complexes are formed by the reaction of NN with interstitial oxygen. Vacancy supersaturation enhances this type of precipitate nucleation. A second nucleation maximum exists around 900 °C. This is assumed to be due to a vacancy assisted oxynitride SiOx Ny based nucleation process. The higher density of the oxynitride phase compared to silicon oxide and a higher residual vacancy concentration would explain the observed shift of the maximum nucleation rate to higher temperatures around 900 °C. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Continuous‐wave and tunable laser operation of Tm:LuVO4 near 1.9 µm under Ti:sapphire and diode laser pumping
Authors: Mateos X. , Liu J. , Zhang H. , Wang J. , Jiang M. , Griebner U. , Petrov V.
Published: February 2006

We demonstrate for the first time to our knowledge laser oscillation of Tm:LuVO4 at room temperature with both Ti:sapphire and diode laser pumping, and tunable operation from 1839 to 1952 nm. Spectroscopic studies indicate higher gain cross section for the σ‐polarization as observed in the laser experiments. (© 2006 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)
Simultaneous determination of tanshinone IIA and its three hydroxylated metabolites by liquid chromatography/tandem mass spectrometry
Authors: Li Peng , Wang Guang‐Ji , Li Jing , Hao Hai‐Ping , Zheng Chao‐Nan
Published: February 2006

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of tanshinone IIA and its three hydroxylated metabolites, tanshinone IIB, hydroxytanshinone IIA and przewaquinone A, in a rat liver microsome was developed and fully validated. A single step of liquid‐liquid extraction with ethyl acetate was utilized in this method. Chromatographic separation of the sample matrix from the analytes and the internal standard diazepam was performed using a Shim‐pack VP‐ODS analytical column. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in selected reaction monitoring (SRM) mode. The method was linear in the concentration range of 1–500 ng/mL for all analytes. The intra‐ and inter‐day precisions (RSD %) were within 15% and deviations of the assay accuracies were within 15.0% for all analytes. The analytes proved to be stable during sample storage, preparation and analyses. This validated method was successfully applied to the enzyme kinetic study of tanshinone IIA in liver microsome. The elimination of tanshinone IIA and formation of tanshinone IIB and hydroxytanshinone IIA in the liver microsome all exhibited a sigmoidal kinetics profile. The formation of przewaquinone A shows a typical hyperbolic profile. In addition, this method has now been applied in the analysis of other bio‐samples including plasma, urine, bile and feces. Copyright © 2006 John Wiley & Sons, Ltd.
Screening for amphetamine and amphetamine‐type drugs in doping analysis by liquid chromatography/mass spectrometry
Authors: Deventer K. , Van Eenoo P. , Delbeke F. T.
Published: February 2006

A selective and sensitive method for the qualitative screening of urine samples for 27 amphetamine and amphetamine‐type drugs in the field of doping analysis is described. The method consists of a liquid‐liquid extraction with diethyl ether at pH 14 and analysis of the extracts with a LCQ‐Deca® mass spectrometer equipped with an atmospheric pressure chemical ionisation interface, operated in positive ionisation mode. The total run time was 15 min. All compounds were analysed in MS2 or MS3. The detection limit for all compounds was lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). Copyright © 2006 John Wiley & Sons, Ltd.
A comparison of the electron ionization and electrospray behaviour of some N,N′‐disubstituted hexahydropyrimidines
Authors: Orelli Liliana R. , García María B. , Perillo Isabel A. , Tonidandel Loris , Traldi Pietro
Published: February 2006

The behaviour of some N,N′‐disubstituted hexahydropyrimidines, a class of naturally occurring compounds of biological and biomedical interest, has been studied in both electron ionization (EI) and electrospray ionization (ESI) modes coupled with collisional experiments (ESI‐MSn). In both techniques, the [M–H]+ ions are highly abundant, even if their formation is generated by two different mechanisms, i.e. H. loss from the M+. species in the case of EI and hydride (H−) abstraction from the molecules in the case of ESI. Furthermore, due to the low, step‐by‐step internal energy deposition typical of collisional experiments performed in an ion trap mass spectrometer, different fragment ions were observed in EI and ESI‐MSn collisions. In both cases, the ions can be related to the original structure and allow us to identify the positions in which the different substituents are present. Copyright © 2006 John Wiley & Sons, Ltd.
Capillary liquid chromatography/atmospheric‐pressure matrix‐assisted laser desorption/ionisation ion trap mass spectrometry: a comparison with liquid chromatography/matrix‐assisted laser desorption/ionisation time‐of‐flight and liquid chromatography/electrospray ionisation quadrupole time‐of‐flight for the identification of tryptic peptides
Authors: Creaser Colin S. , Green Philip S. , Kilby Peter M. , Ratcliffe Lucy
Published: February 2006

The atmospheric‐pressure matrix‐assisted laser desorption/ionisation quadrupole ion trap (AP‐MALDI‐QIT) analysis of tryptic peptides is reported following capillary liquid chromatographic (LC) separation and direct analysis of a protein digest. Peptide fragments were identified by peptide mass fingerprinting from mass spectrometric data and sequence analysis obtained by tandem mass spectrometry of the principal mass spectral peaks using a data‐dependent scanning protocol. These data were compared with those from mass spectrometric analysis using capillary LC/MALDI‐time‐of‐flight (TOF) and capillary LC/electrospray ionisation (ESI)‐quadrupole TOF. For all three configurations the resulting data were searched against the MSDB database, using MASCOT and the sequence coverage compared for each technique. Complementary data were obtained using the three techniques. Copyright © 2006 John Wiley & Sons, Ltd.
Analysis of amitrole by normal‐phase liquid chromatography and tandem mass spectrometry using a sheath liquid electrospray interface
Authors: Girod Marion , Delaurent Corinne , Charles Laurence
Published: February 2006

The coupling of normal‐phase liquid chromatography to tandem mass spectrometry, previously developed in our laboratory, has been applied to the analysis of amitrole. This coupling utilizes an electrospray interface modified to accommodate the introduction of a make‐up solution at the tip of the electrospray probe. A methanolic solution containing 3 mM ammonium acetate delivered at a flow rate of 10 µL · min−1 was found to be the optimal sheath liquid to promote successful ionization of the amitrole. Protonated molecules, arising from in‐source dissociation of ammonium adducts, were subjected to tandem mass spectrometric experiments in a triple‐quadrupole instrument. The main fragmentation reactions were characterized and selected to acquire chromatographic data in the multiple reaction monitoring mode. The limit of detection for amitrole was in the ppm range without any preconcentration step. Enhanced efficiency of ion transmission achievable nowadays in mass spectrometers (this analytical configuration was developed with a 15‐year‐old instrument) is reasonably expected to further improve this detection level. Copyright © 2006 John Wiley & Sons, Ltd.
In vitro metabolism of a new cardioprotective agent, KR‐32570, in human liver microsomes
Authors: Kim Hyojin , Kang Suil , Kim Hyunmi , Yoon Yune‐Jung , Cha Eun‐Young , Lee Hye Suk , Kim Jeong‐Han , Yea Sung Su , Lee Sang‐Seop , Shin Jae‐Gook , Liu Kwang‐Hyeon
Published: February 2006

KR‐32570 (5‐(2‐methoxy‐5‐chlorophenyl)furan‐2‐ylcarbonyl)guanidine) is a new reversible Na+/H+ exchanger inhibitor for preventing ischemia‐reperfusion injury. This study was performed to identify the metabolic pathway of KR‐32570 in human liver microsomes. Human liver microsomal incubation of KR‐32570 in the presence of NADPH and UDPGA resulted in the formation of six metabolites, M1–M6. M1 was identified as O‐desmethyl‐KR‐32570, on the basis of liquid chromatography/tandem mass spectrometric (LC/MS/MS) analysis with the synthesized authentic standard. M2 and M3 were suggested to be hydroxy‐KR‐32570 and hydroxy‐O‐desmethyl‐KR‐32570, respectively. M1, M2, and M3 were further metabolized to their glucuronide conjugates, M4, M5, and M6, respectively. In addition, the specific P450 isoforms responsible for KR‐32570 oxidation to two major metabolites, O‐desmethyl‐KR‐32570 and hydroxy‐KR‐32570, were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 isoforms. The inhibitory potency of KR‐32570 on clinically major P450s was investigated in human liver microsomes. The results show that CYP3A4 contributes to the oxidation of KR‐32570 to hydroxy‐KR‐32570, and CYP1A2 play the predominant role in O‐demethylation of KR‐32570. KR‐32570 was found to inhibit moderately the metabolism of CYP2C8 substrates. Copyright © 2006 John Wiley & Sons, Ltd.
Structural identification of isomers present in technical perfluorooctane sulfonate by tandem mass spectrometry
Authors: Langlois Ingrid , Oehme Michael
Published: February 2006

The structures of isomeric by‐products of technical perfluorooctane sulfonate (PFOS, C8F17SO) were characterized by high‐performance liquid chromatography coupled to mass spectrometry. Ion trap (IT) as well as triple quadrupole (TQ) tandem mass spectrometry (MS/MS) identified ten PFOS isomers with best results being achieved by ion trap MS/MS. In addition to the linear isomer, the main by‐products were isomers with geminal diperfluoromethyl groups and perfluoromethyl‐substituted compounds. Purified fractions of the perfluoromethyl isomers allowed the elucidation of structures and the assignment of the retention times of perfluoroisopropyl‐, 5‐perfluoromethyl‐, 4‐perfluoromethyl‐, 3‐perfluoromethyl‐ and 1‐perfluoromethyl‐PFOS. The concentration of 2‐perfluoromethyl‐PFOS was too low in technical PFOS for a proper identification. Retention times of the geminal substituted diperfluoromethyl isomers could not be properly assigned due to co‐elutions. The number and position of the CF3 groups can be derived from the MS/MS spectra, which allow an unequivocal identification of the isomers. TQ‐MS/MS also allowed us to identify most of the isomers. Copyright © 2006 John Wiley & Sons, Ltd.
Ultra‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry for robust, high‐throughput quantitative analysis of an automated metabolic stability assay, with simultaneous determination of metabolic data
Authors: O'Connor Desmond , Mortishire‐Smith Russell , Morrison Denise , Davies Amy , Dominguez Maria
Published: February 2006

The application of ultra‐performance liquid chromatography coupled to time‐of‐flight mass spectrometry (UPLC/TOFMS) for high‐throughput analysis of a 96‐well plate based metabolic stability assay has been investigated. Full‐scan data were acquired, with run times of 2.5–3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high‐performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound. Copyright © 2006 John Wiley & Sons, Ltd.
From mass to structure: an aromaticity index for high‐resolution mass data of natural organic matter
Authors: Koch B. P. , Dittmar T.
Published: February 2006

Recent progress in Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS) has provided extensive molecular mass data for complex natural organic matter (NOM). Structural information can be deduced solely from the molecular masses for ions with extreme molecular element ratios, in particular low H/C ratios, which are abundant in thermally altered NOM (e.g. black carbon). In this communication we propose a general aromaticity index (AI) and two threshold values as unequivocal criteria for the existence of either aromatic (AI > 0.5) or condensed aromatic structures (AI ≥ 0.67) in NOM. AI can be calculated from molecular formulae which are derived from exact molecular masses of naturally occurring compounds containing C, H, O, N, S and P, and is especially useful for substances with aromatic cores and few alkylations. In order to test the validity of our model index, AI is applied to FTICRMS data of a NOM deep‐water sample from the Weddell Sea (Antarctica), a fulvic acid standard, and an artificial dataset of all theoretically possible molecular formulae. For graphical evaluation a ternary plot is suggested for four‐dimensional data representation. The proposed aromaticity index is a step towards structural identification of NOM and the molecular identification of polyaromatic hydrocarbons in the environment. Copyright © 2006 John Wiley & Sons, Ltd.
Solid‐phase microextraction for gas chromatographic/mass spectrometric analysis of dimethoate in human biological samples
Authors: Gallardo E. , Barroso M. , Margalho C. , Cruz A. , Vieira D. N. , López‐Rivadulla M.
Published: February 2006

A new, simple and rapid procedure for the determination of dimethoate in urine and blood samples was developed using direct immersion solid‐phase microextraction and gas chromatography/mass spectrometry. This technique required only 0.1 mL of sample, and ethion was used as internal standard. Two types of coated fibre were compared (100 µm polydimethylsiloxane, and 65 µm Carbowax™/divinylbenzene). Other parameters, such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH, were optimized to enhance the sensitivity of the method. Limits of detection (LODs) and quantitation (LOQs) were 50 and 100 ng/mL for urine and 200 and 500 ng/mL for blood, respectively. The method was found to be linear between the LOQ and 40 µg/mL for urine, and between the LOQ and 50 µg/mL for blood, with correlation coefficients ranging from 0.9923–0.9996. Precision (intra‐ and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries of dimethoate were 1.24 and 0.50% for urine and blood, respectively. Because of its simplicity and the fact that small volumes of sample are used, the described method can be successfully used in the diagnosis of poisoning by this pesticide, namely in those situations where the sample volume is limited, as frequently occurs in forensic toxicology. Copyright © 2006 John Wiley & Sons, Ltd.
Screening for metabolically stable aryl‐propionamide‐derived selective androgen receptor modulators for doping control purposes
Authors: Thevis Mario , Kamber Matthias , Schänzer Wilhelm
Published: February 2006

Anabolic agents have been among the most frequently detected drugs in amateur and professional sport. A novel class of therapeutics presumably complementing anabolic steroids in the near future includes so‐called selective androgen receptor modulators (SARMs) that have been under clinical investigations for several years. Although not yet commercially available, their potential for misuse in sports is high. Four aryl‐propionamide‐derived SARMs were synthesized in order to establish a fast and robust screening procedure using liquid chromatography/electrospray ionization tandem mass spectrometry. Synthesized compounds were characterized by high‐resolution/high‐accuracy mass analysis employing a linear ion trap‐Orbitrap hybrid mass spectrometer while routine analyses were conducted on a triple‐quadrupole mass spectrometer. Characteristic product ions obtained by collision‐induced dissociation were found at m/z 289 and 261 as well as m/z 269 and 241 representing the bisubstituted aniline residues of selected model compounds. Assay validation was performed regarding lower limit of detection (1 ng/mL), recovery (85–105%), intraday precision (7.6–11.6%) and interday precision (9.9–14.4%), and precursor ion scan experiments on diagnostic product ions enabled the detection of a structurally related compound at 50 ng/mL. Copyright © 2006 John Wiley & Sons, Ltd.
Matrix‐assisted laser desorption/ionization in‐source decay combined with tandem time‐of‐flight mass spectrometry of permethylated oligosaccharides: targeted characterization of specific parts of the glycan structure
Authors: Wuhrer Manfred , Deelder André M.
Published: February 2006

Matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometry (MALDI‐TOF/TOF‐MS) has been introduced in recent years as a valuable tool for the structural characterization of permethylated oligosaccharides. In this report, we describe the combination of MALDI in‐source decay (ISD) with the subsequent TOF/TOF‐MS analyses of specific fragments, allowing the detailed characterization of the selected part of the oligosaccharide molecule. Part of the second‐generation fragment ions were different from those observed in conventional MALDI‐TOF/TOF‐MS experiments. Other fragments, which had already been observed in conventional MALDI‐TOF/TOF‐MS and again showed up in second‐generation fragment analysis, could be assigned to specific parts of the molecule. Our approach disclosed different structural features of the oligosaccharides: due to permethylation, the glycosidic linkage fragments allowed the distinction between terminal, monosubstituted and disubstituted monosaccharides and indicated the oligosaccharide sequence. Moreover, substitution positions were deduced based on characteristic cross‐ring fragmentation by high‐energy collision‐induced fragmentation. In conclusion, combination of MALDI‐ISD with TOF/TOF‐MS allows the detailed characterization of specific moieties of permethylated oligosaccharides and is, therefore, a powerful technique for structural glycomics. Copyright © 2006 John Wiley & Sons, Ltd.
Targeted comparative proteomics by liquid chromatography/matrix‐assisted laser desorption/ionization triple‐quadrupole mass spectrometry
Authors: Melanson Jeremy E. , Chisholm Kenneth A. , Pinto Devanand M.
Published: February 2006

Here we report the first application of a matrix‐assisted laser desorption/ionization (MALDI) triple‐quadrupole mass spectrometer for targeted proteomics. Employing an amine‐specific isotopic labelling approach, the technique was validated using five randomly selected bovine serum albumin peptides differentially labelled at known ratios. An indirect benefit of the isotopic labelling technique is a significant enhancement of the a1 ion in tandem mass (MS/MS) spectra of all peptides studied. Therefore, the a1 ion was selected as the fragment ion for multiple reaction monitoring (MRM) in all cases, eliminating tedious method development and optimization. Accurate quantification was achieved with an average relative standard deviation (RSD) of 5% (n = 5) and a detection limit of 14 amol. The technique was then applied to validate an important virulence biomarker of the fungal pathogen Candida albicans, which was not accurately quantified using global proteomics experiment employing two‐dimensional liquid chromatography/electrospray ionization tandem mass spectrometry (2D‐LC/ESI)‐MS/MS. Using LC/MALDI‐MRM analysis of five tryptic peptides, the protein PHR1 was found to be upregulated in the hyphal (pathogenic) form of C. albicans by a factor of 7.7 ± 0.8. Copyright © 2006 John Wiley & Sons, Ltd.
Liquid chromatography/tandem mass spectrometry characterization of oxidized amyloid beta peptides as potential biomarkers of Alzheimer's disease
Authors: Inoue Koichi , Garner Carlos , Ackermann Bradley L. , Oe Tomoyuki , Blair Ian A.
Published: February 2006

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid β peptides. There is substantial evidence that amyloid β is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal‐catalyzed oxidation can mimic in vivo oxidation of amyloid β because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal‐catalyzed oxidation occurs on histidines‐13 and ‐14. Unfortunately, the amyloid β peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid β1–16 and amyloid β1–40 resulted in the formation of tryptic peptides corresponding to amyloid β6–16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine‐13 and histidine‐14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid β6–16 tryptic fragments derived from full‐length amyloid β peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine‐35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress. Copyright © 2006 John Wiley & Sons, Ltd.
Matrix‐assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue
Authors: Hsieh Yunsheng , Casale Roger , Fukuda Elaine , Chen Jiwen , Knemeyer Ian , Wingate Julia , Morrison Richard , Korfmacher Walter
Published: February 2006

Matrix‐assisted laser desorption/ionization hyphenated with quadrupole time‐of‐flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI‐QTOF following a direct infusion of 3H‐clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high‐performance liquid chromatography/tandem mass spectrometry (HPLC‐MS/MS) procedures. Copyright © 2006 John Wiley & Sons, Ltd.