bluuebunny/crossref_metadata_2025 huggingface dataset:
Facile preparation of titanium phosphate‐modified chitosan for selective capture of phosphopeptides
Authors: Shen Feng , Hu Yufeng , Guan Ping , Ren Xueqin
Published: December 2012

A facile two‐step method for preparing chitosan‐based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan‐based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α‐casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum‐loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α‐casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α‐casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt‐stressed Arabidopsis thaliana leaves.
Optimization of hydrophilic interaction LC by univariate and multivariate methods and its combination with salting‐out liquid–liquid extraction for the determination of antihypertensive drugs in the environmental waters
Authors: Wang Qing , Yin Chen‐ru , Xu Li
Published: December 2012

Hydrophilic interaction LC for the separation of four antihypertensive drugs was optimized by both univariate and multivariate methods. The column efficiency, resolution, and separation time were used as the three assessment parameters. The best separation condition of 97% ACN with 3% aqueous buffer containing 50 mM ammonium acetate at a pH of 3.0 was obtained by the two optimization methods. The multivariate optimization, orthogonal array design herein, was demonstrated to be a little tedious, but afforded a much better understanding of underlying separation factors. The content of ACN in the mobile phase contributed most significantly to separation. Furthermore, sample diluent and injection volume were found to influence the chromatographic performance. To match the hydrophilic interaction LC mobile phase, a proper sample pretreatment method, salting‐out liquid–liquid extraction, in which ACN was the extractant, was chosen. Since reserpine was unstable under both acidic and alkaline conditions, it was not studied in this part. The optimal salting‐out liquid–liquid extraction parameters were as follows: 400 μL ACN was added to 1 mL sample solution containing 500 mg NH4Cl at a pH of 14.0. The linearity ranged from 0.01 to 1.00 μg/mL with r2 > 0.9937. The LODs were between 1.9 and 2.5 ng/mL. The developed method was applied to the environmental water sample with good performance.
Rapid determination of α‐tocopherol in cereal grains using dispersive liquid–liquid microextraction followed by HPLC
Authors: Liu Shuhui , Xie Qilong , Cao Jiangping , Song Pingping , Chen Jie , Bai Weiwei
Published: December 2012

The traditional for the determination of α‐tocopherol in cereal grains includes saponification of a sample followed by liquid–liquid extraction, and it is time‐ and solvent consuming. In this study, a dispersive liquid–liquid microextraction (DLLME) method was developed to extract α‐tocopherol in situ from the saponified grain sample solution. The DLLME experimental parameters including the type and volume of extractants, the volume of dispersers, the addition of salt and the extraction/centrifuging time were examined and optimized. The recommended analytical procedure showed excellent precision (relative SDs of the α‐tocopherol amount of 3.1% over intraday and 7.2% over interday), high sensitivity (the detection limit of 1.9 ng/mL), and strong recovery values (88.9–102.5%). In addition, statistical analyses showed no significant difference between the detected amounts of α‐tocopherol found by the standardized method and this new procedure. The method was successfully applied to determining the amounts and distribution of α‐tocopherol in 14 cereal grain samples.
Symptoms in chronic rhinosinusitis with and without nasal polyps
Authors: Dietz de Loos Dirk A. E. , Hopkins Claire , Fokkens Wytske J.
Published: December 2012

Objectives/Hypothesis:

In this study we analyzed differences in symptoms scored between chronic rhinosinusitis patients with (CRSwNP) and without nasal polyps (CRSsNP). According to the European Position Paper on Rhinosinusitis and Nasal Polyps, CRSwNP and CRSsNP diagnoses are defined by clinical criteria supported with endoscopy. We wanted to know if it is possible to make an accurate distinction between patients with and without nasal polyps based on clinical impression.

Study Design:

Retrospective case‐control study.

Methods:

We collected Rhinosinusitis Outcome Measure 31 (RSOM‐31) questionnaires from chronic rhinosinusitis patients with and without nasal polyps and compared mean total RSOM‐31 scores, mean domain scores, mean symptoms scores, and percentages of patients reporting symptoms per diagnosis based on endoscopy and computed tomography scan.

Results:

RSOM‐31 questionnaires were collected from 234 patients. Although the total RSOM‐31 score was similar and symptomatology considerably overlapping, patients with CRSwNP scored significantly higher and more often on nasal symptoms such as “rhinorrhea” and “decreased sense of taste or smell.” Patients with CRSsNP significantly scored more often and higher on “facial pain” and “ear pain.”

Conclusions:

Although there were significant differences in scores on several symptoms, there was considerable overlap of many symptoms, and it remains difficult to distinguish between CRSwNP and CRSsNP based on clinical impression alone. Laryngoscope, 2013
The exopolysaccharide gene cluster Bcam1330–Bcam1341 is involved in Burkholderia cenocepacia biofilm formation, and its expression is regulated by c‐di‐GMP and Bcam1349
Authors: Fazli Mustafa , McCarthy Yvonne , Givskov Michael , Ryan Robert P. , Tolker‐Nielsen Tim
Published: December 2012

In Burkholderia cenocepacia, the second messenger cyclic diguanosine monophosphate (c‐di‐GMP) has previously been shown to positively regulate biofilm formation and the expression of cellulose and type‐I fimbriae genes through binding to the transcriptional regulator Bcam1349. Here, we provide evidence that cellulose and type‐I fimbriae are not involved in B. cenocepacia biofilm formation in flow chambers, and we identify a novel Bcam1349/c‐di‐GMP‐regulated exopolysaccharide gene cluster which is essential for B. cenocepacia biofilm formation. Overproduction of Bcam1349 in trans promotes wrinkly colony morphology, pellicle, and biofilm formation in B. cenocepacia. A screen for transposon mutants unable to respond to the overproduction of Bcam1349 led to the identification of a 12‐gene cluster, Bcam1330–Bcam1341, the products of which appear to be involved in the production of a putative biofilm matrix exopolysaccharide and to be essential for flow‐chamber biofilm formation. We demonstrate that Bcam1349 binds to the promoter region of genes in the Bcam1330–Bcam1341 cluster and that this binding is enhanced by the presence of c‐di‐GMP. Furthermore, we demonstrate that overproduction of both c‐di‐GMP and Bcam1349 leads to increased transcription of these genes, indicating that c‐di‐GMP and Bcam1349 functions together in regulating exopolysaccharide production from the Bcam1330–Bcam1341 gene cluster. Our results suggest that the product encoded by the Bcam1330–Bcam1341 gene cluster is a major exopolysaccharide that provides structural stability to the biofilms formed by B. cenocepacia, and that its production is regulated by c‐di‐GMP through binding to and promotion of the activity of the transcriptional regulator Bcam1349.
Analysis of CYP2D6 genotype and response to tetrabenazine
Authors: Mehanna Raja , Hunter Christine , Davidson Anthony , Jimenez‐Shahed Joohi , Jankovic Joseph
Published: December 2012

Tetrabenazine is effective in the treatment of the chorea associated with Huntington disease and other hyperkinetic movement disorders. Following oral administration, tetrabenazine is hepatically transformed into 2 active metabolites that are CYP2D6 substrates. There are 4 CYP2D6 genotypes: poor metabolizers, intermediate metabolizers, extensive metabolizers, and ultrarapid metabolizers. CYP2D6 genotyping was performed on sequential subjects treated with tetrabenazine, but results were not known at the time of titration. Duration of titration to a stable dose, total daily dose, response rating scores, and adverse events were retrospectively collected and subsequently analyzed. Of 127 patients, the majority (n = 100) were categorized as extensive metabolizers, 14 as intermediate metabolizers, 11 as poor metabolizers, and 2 as ultrarapid metabolizers. Ultrarapid metabolizer patients needed a longer titration (8 vs 3.3, 4.4, and 3 weeks, respectively; P < .01) to achieve optimal benefit and required a higher average daily dose than the other patients, but this difference did not reach statistical significance. The treatment response was less robust in the intermediate metabolizer group when compared with the extensive metabolizer patients (P = .013), but there were no statistically significant differences between the various groups with regard to adverse effects. Our findings demonstrate that, aside from the need for a longer titration in the ultrarapid metabolizers, there are no distinguishing features of patients with various CYP2D6 genotypes, and therefore the current recommendation to systematically genotype all patients prescribed more than 50 mg/day of tetrabenazine should be reconsidered. © 2012 Movement Disorder Society
White light spectroscopy for free flap monitoring
Authors: Fox Paige M. , Zeidler Kamakshi , Carey Joseph , Lee Gordon K.
Published: December 2012

White light spectroscopy non‐invasively measures hemoglobin saturation at the capillary level rendering an end‐organ measurement of perfusion. We hypothesized this technology could be used after microvascular surgery to allow for early detection of ischemia and thrombosis. The Spectros T‐Stat monitoring device, which utilizes white light spectroscopy, was compared with traditional flap monitoring techniques including pencil Doppler and clinical exam. Data were prospectively collected and analyzed. Results from 31 flaps revealed a normal capillary hemoglobin saturation of 40–75% with increase in saturation during the early postoperative period. One flap required return to the operating room 12 hours after microvascular anastomosis. The T‐stat system recorded an acute decrease in saturation from ∼50% to less than 30% 50 min prior to identification by clinical exam. Prompt treatment resulted in flap salvage. The Spectros T‐Stat monitor may be a useful adjunct for free flap monitoring providing continuous, accurate perfusion assessment postoperatively. © 2012 Wiley Periodicals, Inc. Microsurgery, 2013.
Active management of selective intrauterine growth restriction with abnormal Doppler in monochorionic diamniotic twin pregnancies diagnosed in the second trimester of pregnancy
Authors: Chalouhi G. E. , Marangoni M. A. , Quibel T. , Deloison B. , Benzina N. , Essaoui M. , Al Ibrahim A. , Stirnemann J. J. , Salomon L. J. , Ville Y.
Published: December 2012

Objective

This study aims to compare outcomes of active management of monochorionic diamniotic twin pregnancies complicated with severe intrauterine growth restriction (IUGR) of one twin before 24 weeks with continuous or intermittent absent or reversed end‐diastolic flow (AREDF) in the umbilical artery, with or without twin‐to‐twin transfusion syndrome (TTTS).

Method

This study is a retrospective comparison of 45 consecutive cases of severe selective IUGR (s‐IUGR) defined as an estimated fetal weight at or below the fifth centile with a >25% weight discordance and AREDF in the umbilical artery before 24 weeks and 166 consecutive cases of TTTS stage III, with AREDF in the donor (TTTS3D) and also with s‐IUGR. These were treated by either selective laser photocoagulation of chorionic vessels (SLPCV) or cord coagulation (CC).

Results

The 166 cases of TTTS3D were treated by SLPCV, whereas 23 and 22 cases of s‐IUGR were treated by SLPCV and CC, respectively. Overall survival was 52.17% or 45.45% in s‐IUGR treated by SLPCV or CC, respectively, and 48.49% in TTTS3D. The survival of appropriately grown for gestational age (AGA) twins following CC (90.9%) was higher than that following SLPCV in s‐IUGR (74%) or in recipient twins of TTTS3D (55.42%) (p = 0.001). Survival of the IUGR twin was 30% and 41.56% with SLPCV in s‐IUGR and TTTS3D, respectively.

Conclusions

Active management of severe IUGR with AREDF in the umbilical artery seems beneficial. Survival rates with SLPCV were similar in s‐IUGR and TTTS3D. However, there was a trend for higher survival rates in the AGA twin for CC. The choice of the technique should be driven by objective counseling on survival of both IUGR and AGA twins and therefore by the utility‐based ethical values expressed by the pregnant woman. © 2012 John Wiley & Sons, Ltd.
Circulating mRNA for epidermal growth factor‐like domain 7 (EGFL7) in maternal blood and early intrauterine growth restriction. A preliminary analysis
Authors: Zanello Margherita , DeSanctis Paola , Pula Giulia , Zucchini Cinzia , Pittalis Maria Carla , Rizzo Nicola , Farina Antonio
Published: December 2012

Objective

To evaluate the alteration in epidermal growth factor‐like domain 7 (EGFL7) mRNA expression in maternal blood from pregnancies affected by early‐onset intrauterine growth restriction (IUGR) at 20–24 weeks.

Method

Case–control study encompassing six women with pregnancies affected by IUGR (cases) matched in a 1 : 7 ratio for gestational age and fetal gender with 42 controls. We quantified EGFL7 mRNA expression in normal and IUGR patients. Matched rank–sum analysis and multiples of median were used to evaluate differences of the marker of interest between cases and controls. Spearman regression analysis was used to correlate the estimated fetal weight at blood sampling with the EGFL7 mRNA values.

Results

The mean observed rank in the IUGR group was significantly higher than that of controls (6.67 vs 4.19, p = 0.01). Pregnancies affected with IUGR exhibited 1.70‐fold higher levels of maternal EGFL7 mRNA compared with matched controls (p = 0.014). EGFL7 mRNA values were inversely correlated with estimated fetal weight (Spearman's ρ = −0.429, p = 0.198).

Conclusion

Early IUGR at 20–24 weeks’ gestation is associated with higher values of EGFL7 expression in maternal plasma. © 2012 John Wiley & Sons, Ltd.
Different indications, warnings and precautions, and contraindications for the same drug—an international comparison of prescribing information for commonly used psychiatric drugs
Authors: Pfistermeister Barbara , Schenk Christian , Kornhuber Johannes , Bürkle Thomas , Fromm Martin F , Maas Renke
Published: December 2012

Purpose

We aim to derive an internationally applicable data set to improve prescription safety of psychiatric drugs.

Methods

We performed an in‐depth analysis of the concordance of prescribing information of 10 key psychiatric drugs across four major drug markets with regard to indications, warnings and precautions, and contraindications.

Results

The individual prescribing information covered on average 71.4 ± 30.3% of all named indications, 59.5 ± 17.1% of all potential warnings and precautions and 70.1 ± 24.4% of all applicable contraindications.

Conclusion

This substantial variation in key prescribing information across countries highlights the need for a better international cooperation and standardization of prescribing information. Copyright © 2012 John Wiley & Sons, Ltd.
CORRELATES OF PURE AND CO‐OCCURRING PROACTIVE AND REACTIVE AGGRESSORS IN HONG KONG
Authors: Chan Jacob Yuichung , Fung Annis L. , Gerstein Lawrence H.
Published: December 2012

Research on aggression in the West has focused mainly on the dichotomy between proactive and reactive aggression, but not the co‐occurring proactive–reactive aggression subtype, despite its prevalence. The authors investigated the differences in psychological and behavioral correlates among proactive, reactive, and proactive–reactive student aggressors and nonaggressors in Hong Kong, China, based on their scores on the Reactive and Proactive Aggression Questionnaire. Participants were 1,356 secondary school students (805 males and 551 females, aged 11 to 18 years). A one‐way multivariate analysis of covariance was conducted to examine group differences in aggression, attention deficits, anxiety/depression, and delinquency. Results showed that proactive–reactive aggressors were significantly more aggressive, less attentive, more anxious/depressed, and more engaged in delinquent behaviors. Reactive and proactive aggressors did not differ in three of these four behaviors. Implications for research and practice when working with proactive–reactive aggressors are discussed.
Hyperbranched aliphatic polyether polyols
Authors: Schömer Martina , Schüll Christoph , Frey Holger
Published: December 2012

Hyperbranched polymers, dendritic macromolecules with branch‐on‐branch structures, have become an important polymer class since the early 1990s. They combine several advantages of the perfectly branched dendrimers with easy accessibility, typically in a one‐step synthesis. Hyperbranched polyethers are a particularly interesting class of chemically stable and often biocompatible materials. Multifunctional hyperbranched polyethers with controllable molar mass and comparably low polydispersities can been prepared using hydroxyl‐functional epoxides or oxetanes for polymerization via anionic and cationic polymerization mechanisms. Here, we review the progress in the preparation, characterization, and application of these uniquely versatile aliphatic polyether polyols. Their unusual mechanical, thermal, and solution properties render them useful for a variety of applications, for example, as building blocks for various complex macromolecular architectures or in biomedical applications. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013
High dielectric performance of tactic polynorbornene derivatives synthesized by ring‐opening metathesis polymerization
Authors: You Zewang , Gao Danyi , Jin Ouyue , He Xiaohua , Xie Meiran
Published: December 2012

Functional polynorbornenes (PNBEs) containing pyrrolidine moiety and bis(trifluoromethyl)biphenyl side group were synthesized via ring‐opening metathesis polymerization (ROMP), and the microstructure of polymer chain was characterized by NMR spectroscopy. Poly(N‐3,5‐bis(trifluoromethyl)biphenyl‐norbornene‐pyrrolidine) (PTNP) and poly(N‐phenyl‐norbornene‐pyrrolidine) (PPNP) are supposed to have practically trans double bonds and adopt isotactic syn conformation, whereas poly(N‐3,5‐bis(trifluoromethyl)biphenyl‐norbornene‐dicarboximide) (PTNDI) has both trans and cis double bonds and atactic microstructure. PTNP, PTNDI, and PPNP have much different dielectric constants of 20, 7, and 3, respectively, which is attributed to both the polar 3,5‐bis(trifluoromethyl)biphenyl group and the stereoregular chain structure. The existence of rigid pyrrolidine moiety has a positive contribution to form the tactic polymer chain during ROMP. Polymers are highly thermal stable up to ∼300 °C. Having good dielectric properties and thermal stability, these functional PNBEs are expected as the potential dielectric material in thin film capacitors. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013
Effects of the acceptor conjugation length and composition on the electrical memory characteristics of random copolyimides
Authors: Kurosawa Tadanori , Lai Yi‐Cang , Yu An‐Dih , Wu Hung‐Chin , Higashihara Tomoya , Ueda Mitsuru , Chen Wen‐Chang
Published: December 2012

Resistive‐switching memories based on copolyimides (coPIs), PI‐NTCDIX and PI‐BTCDIX, with different compositions of 4,4′‐diamino‐4″‐methyltriphenylamine (AMTPA), 4,4′‐(hexafluoroisopropylidene)diphthalic anhydride, and N,N′‐bis‐(4‐aminophenyl)‐1,8:4,5‐naphthalenetetracarboxydiimide (NTCDI) or N,N′‐bis‐(4‐aminophenyl)‐1,2:4,5‐benzenetetracarboxydiimide (BTCDI) have been developed. By varying the feed ratio of monomers, PI‐NTCDIX and PI‐BTCDIX showed tunable optical and electronic properties through the charge transfer (CT) between AMTPA and NTCDI or BTCDI. The memory devices based on PI‐NTCDIX exhibited the tunable electrical bistability from the volatile dynamic random access memory to nonvolatile write once read many memory characteristics as the NTCDI composition increased. The OFF/ON electrical switching transition was mainly attributed to the CT mechanism for the charge separated high conductance, based on the analysis of model compounds and density functional theory calculation. Also, the volatility of the memory device depended on the stability of CT complex. The long conjugation and high electron affinity of the NTCDI moiety stabilized the radical anion generated in the CT complex and prevented the recombination of segregated radical species even through applying the high positive or negative voltage. On the other hand, the memory devices based on PI‐BTCDIX showed a rather unique behavior compared with those based on PI‐NTCDIX. At the low BTCDI composition, the device exhibited volatile memory property. However, no switching behavior was observed at the high BTCDI composition due to the low highest occupied molecular orbital energy level of BTCDI. Combining these results and our previous study on perylenebisimide (PBI), we concluded that memory characteristics could be tailored by changing the conjugation length (PBI > NTCDI > BTCDI) and the acceptor composition in random coPIs. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013
An enhanced host–guest electro‐optical polymer system using poly(norbornene‐dicarboximides) via ROMP
Authors: Yu Feng , Spring Andrew M. , Li Lu , Qiu Feng , Yamamoto Kazuhiro , Maeda Daisuke , Ozawa Masaaki , Odoi Keisuke , Yokoyama Shiyoshi
Published: December 2012

High glass transition temperature poly(N‐cyclohexyl‐5‐norbornene‐2,3‐dicarboximide)s (NDI)s prepared by ring opening metathesis polymerization yielded polymers with a narrow polydispersity and well‐controlled molecular weight materials when using the Grubbs first generation initiator. Polymers produced using the Grubbs second generation initiator could not be controlled easily. By initiator selection it was also possible to synthesize polymers with either 98 or 52% trans microstructures. These materials were employed as electro‐optic (EO) polymer hosts for high molecular hyperpolarizability (β) phenyl vinylene thiophene vinylene bridge chromophores. This chromophore was modified by the incorporation of a tert‐butyldiphenylsilane group. The addition was able to further increase its EO coefficient (r33) to reach 93 pm/V in a trans rich poly(NDI) produced by the Grubbs first generation initiator, compared to a benchmark chromophore / polymer combination. We investigated in detail the relationship between polymer microstructure and their absolute molecular weight on forming the best host–guest with the high β chromophore. Our results indicate that by utilizing a very simple host–guest system a high r33 can be realized. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013
Materials for the preparation of polymer pen lithography tip arrays and a comparison of their printing properties
Authors: Zhong Xiao , Bailey Nevette A. , Schesing Kevin B. , Bian Shudan , Campos Luis M. , Braunschweig Adam B.
Published: December 2012

Polymer Pen Lithography (PPL) uses an array of polymeric tips, typically composed of poly(dimethyl siloxane), to transfer ink onto a surface and create patterns of soft molecules with micrometer to nanometer feature dimensions. In this study, tip arrays were fabricated from poly(methyl methacrylate), poly([methyl methacrylate]‐co‐[butyl methacrylate]), and poly(3‐mercaptopropylmethylsiloxane), and used to pattern 1‐mercaptohexadecanoic acid onto Au surfaces to determine the fidelity of pattern transfer by PPL as a function of the mechanical properties of the materials. It was found that the dependence between the applied force and feature edge length correlates directly to the mechanical properties of each of the polymers used to fabricate the tip arrays, where stiffer polymers have a reduced dependence between the applied force and feature size. This study demonstrates that PPL tip arrays can be composed of a wide variety of materials whose choice is determined by the desired printing application. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013
Insulin analog with additional disulfide bond has increased stability and preserved activity
Authors: Vinther Tine N. , Norrman Mathias , Ribel Ulla , Huus Kasper , Schlein Morten , Steensgaard Dorte B. , Pedersen Thomas Å. , Pettersson Ingrid , Ludvigsen Svend , Kjeldsen Thomas , Jensen Knud J. , Hubálek František
Published: December 2012

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα‐Cα distances, solvent exposure, and side‐chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C‐terminus of the B‐chain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild‐type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R‐state conformation and thus showing that the R‐state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.
Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts
Authors: Yang Chunyan , Pan Huirong , Wei Minxi , Zhang Xiao , Wang Nan , Gu Ying , Du Hailian , Zhang Jun , Li Shaowei , Xia Ningshao
Published: December 2012

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein–protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368–606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH4)2SO4], sodium sulfate (Na2SO4), sodium chloride (NaCl), and ammonium chloride (NH4Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH4)2SO4 was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28‐aa region (aa368–395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu372, Leu375, and Leu395of p239 was found to be critical for particle assembly, which was revealed by site‐directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.
A simple criterion for gas chromatography/mass spectrometric analysis of thermally unstable compounds, and reassessment of the by‐products of alkyl diazoacetate synthesis
Authors: Kornilova Tatiana A. , Ukolov Anton I. , Kostikov Rafael R. , Zenkevich Igor G.
Published: December 2012

RATIONALE

A principal limitation of gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) is the thermal instability of analytes. We propose that the injector and column temperatures should not exceed the atmospheric pressure boiling point, without decomposition, of the highest homologue of the series being analyzed, instead of the time‐consuming procedure of obtaining chromatograms using different temperatures.

METHODS

A series of thermally unstable diazocarbonyl compounds, alkyl diazoacetates (predicted limit of stability approx. 140 °C, the boiling point of ethyl diazoacetate), was selected for GC/MS analysis using standard equipment. Different GC separation conditions were selected so that the retention temperatures of target compounds were both below and above 140 °C.

RESULTS

Analyzing alkyl diazoacetates within their thermal stability range permitted reanalysis of their typical synthesis by‐products. No dialkyl fumarate or maleate impurities, principal decomposition products which have often been reported previously, were found. Instead, alkyl esters of glycolic acid nitrate, O2NOCH2CO2R, and 'pseudo‐dimeric' products, ROCO[C2H3NO]CO2R, were discovered for the first time.

CONCLUSIONS

Avoiding the decomposition of thermally unstable organic compounds during GC and/or GC/MS analysis requires estimating their degradation temperature limits. This limit can be estimated as being equal to the atmospheric pressure boiling point of the highest homologue in the homologous series under consideration that does not decompose on boiling. Copyright © 2012 John Wiley & Sons, Ltd.
High‐throughput liquid chromatography/mass spectrometry method for the quantitation of small molecules using accurate mass technologies in supporting discovery drug screening
Authors: Ding Xiao , Ghobarah Hesham , Zhang Xiaolin , Jaochico Allan , Liu Xingrong , Deshmukh Gauri , Liederer Bianca M. , Hop Cornelis E. C. A. , Dean Brian
Published: December 2012

RATIONALE

Drug discovery samples are routinely analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods on triple quadrupole mass spectrometers employing multiple reaction monitoring (MRM). In order to improve analysis throughput, quantitation of small molecules on a quadrupole time‐of‐flight (QqTOF) instrument using TOF scan and high‐resolution MRM (MRM‐HR) modes was evaluated in this study.

METHODS

Cassette dosed plasma and brain samples from nine compounds were extracted using a protein precipitation method. Separation was achieved by reversed‐phase liquid chromatography. Mass spectrometric analysis was performed using TOF scan and high‐resolution MRM approaches on a QqTOF mass spectrometer with turbo‐ionspray ionization. Results were compared to those obtained on a triple quadrupole mass spectrometer.

RESULTS

The dynamic range varied depending on compounds and instruments and was similar between the MRM on QqQ and full TOF scan mode on QqTOF. Linear or quadratic regression and 1/x2 weighting were used. Resolution on the QqTOF instrument was around 32000 and mass accuracy was within 4.4 ppm. The MRM‐HR method showed better sensitivity compared to the TOF scan method, and was comparable to the MRM on a QqQ mass spectrometer. Assay accuracy was within ±25%.

CONCLUSIONS

A TOF scan method allowed the use of the generic method without compound‐specific optimization and was an alternative choice for routine high‐throughput quantitation of small molecules. The MRM‐HR method on the QqTOF showed good sensitivity which was comparable to that obtained by the MRM method on the triple quadrupole mass spectrometer. Copyright © 2012 John Wiley & Sons, Ltd.
Combination of Edman degradation of peptides with liquid chromatography/mass spectrometry workflow for peptide identification in bottom‐up proteomics
Authors: Lobas Anna A. , Verenchikov Anatoly N. , Goloborodko Anton A. , Levitsky Lev I. , Gorshkov Mikhail V.
Published: December 2012

RATIONALE

High‐throughput methods of proteomics are essential for identification of proteins in a cell or tissue under certain conditions. Most of these methods require tandem mass spectrometry (MS/MS). A multidimensional approach including predictive chromatography and partial chemical degradation could be a valuable alternative and/or addition to MS/MS.

METHODS

In the proposed strategy peptides are identified in a three‐dimensional (3D) search space consisting of retention time (RT), mass, and reduced mass after one‐step partial Edman degradation. The strategy was evaluated in silico for two databases: baker's yeast and human proteins. Rates of unambiguous identifications were estimated for mass accuracies from 0.001 to 0.05 Da and RT prediction accuracies from 0.1 to 5 min. Rates of Edman reactions were measured for test peptides.

RESULTS

A 3D description of proteolytic peptides allowing unambiguous identification without employing MS/MS of up to 95% and 80% of tryptic peptides from the yeast and human proteomes, respectively, was considered. Further extension of the search space to a four‐dimensional one by incorporating the second N‐terminal amino acid residue as the fourth dimension was also considered and was shown to result in up to 90% of human peptides being identified unambiguously.

CONCLUSIONS

The proposed 3D search space can be a useful alternative to MS/MS‐based peptide identification approach. Experimental implementations of the proposed method within the on‐line liquid chromatography/mass spectrometry (LC/MS) and off‐line matrix‐assisted laser desorption/ionization (MALDI) workflows are in progress. Copyright © 2012 John Wiley & Sons, Ltd.
A comparison of pretreatment methods for the analysis of phosphate oxygen isotope ratios in bioapatite
Authors: Grimes Vaughan , Pellegrini Maura
Published: December 2012

RATIONALE

The integrity of the biological phosphate oxygen isotope (δ18Op) signal is thought to be contingent upon the complete removal of competing sources of oxygen such as associated organic matter. A range of pretreatment methods to purify phosphate material from competing sources of oxygen has been reported, with contradictory evidence on the usefulness and efficiency of one or another. Yet, a systematic comparison of these techniques for bioapatite phosphate has not been conducted.

METHODS

Chemical and thermal pretreatment techniques were tested for their effectiveness at removing organic matter and the likelihood that they modify original δ18O values. The test was performed in inorganic (synthetic apatite and a phosphorite rock) and organic (bone and tooth tissues) phosphate materials for which we had an expectation of the actual original δ18Op value. Analysis of nitrogen content (wt.%), scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy were employed.

RESULTS

We detected variable efficiency at removing organic matter between pretreatment methods with no correlation to any specific structural change. The δ18Op results showed considerable variation between samples pretreated with the different methods and the untreated samples, with a compositional range of up to 4.5 ‰ in the bone samples. Variations of the δ18Op values within error were found for tooth enamel, phosphorite rock and inorganic apatite.

CONCLUSIONS

We recommend a cautious approach when interpreting and comparing δ18Op data from bone samples treated with different pretreatment protocols. In general, the untreated samples seem to show δ18Op values closer to the expected ones. According to our results, pretreatment is completely unnecessary in highly mineralized tissues. Copyright © 2012 John Wiley & Sons, Ltd.
Analytical challenges in the quantitative determination of 2H/1H ratios of methyl iodide
Authors: Feakins Sarah J. , Rincon Miguel , Pinedo Paulina
Published: December 2012

RATIONALE

A published method to determine the hydrogen isotope (2H/1H) ratios of methoxyl groups, cleaved from lignin by hydroiodic acid (HI), calls for the analysis of methyl iodide (CH3I). However, analysis of halogenated compounds by gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS) yields incomplete conversion into H2 and unwanted HX products post‐pyrolysis.

METHODS

We tested analytical capabilities when measuring a pure CH3I standard using a GC Isolink pyrolysis furnace at 1420 °C and a DELTA VPlus™ isotope ratio mass spectrometer, both by Thermo Scientific. We added and tested the efficacy of either a secondary reduction furnace containing heated metals or a cold trap to eliminate unwanted HI prior to IRMS analysis for 2H/1H determinations.

RESULTS

While 94% of the hydrogen in CH3I is converted into H2 in GC/P/IRMS, 6% yields HI. The use of either a secondary reduction furnace or cold trap can eliminate HI. However, secondary reduction is untenable given changes in reduction/transmission efficiency and 2H/1H drift. A cold trap provides a pragmatic solution: the δ2H values of −97 ‰ were measured from injections of 23–57 µg of CH3I to a precision of 4.5 ‰ (n = 26), within the error of the offline measurements.

CONCLUSIONS

We have refined an earlier method to measure the δ2H values of CH3I by GC/P/IRMS by adding a cold trap to remove HI. This approach should facilitate future experimentation with the measurement of CH3I liberated from natural samples for potentially diverse applications including forensic sourcing of wood and tree‐wood‐based reconstructions of source water. Copyright © 2012 John Wiley & Sons, Ltd.
Direct analysis in real time high‐resolution mass spectrometry for high‐throughput analysis of antiparasitic veterinary drugs in feed and food
Authors: Martínez‐Villalba Anna , Vaclavik Lukas , Moyano Encarnación , Galceran Maria Teresa , Hajslova Jana
Published: December 2012

RATIONALE

Direct analysis in real time (DART) is a novel ionization technique that has been demonstrated in numerous applications as a useful tool for fast and convenient mass spectrometry (MS)‐based analysis of complex samples. In this study, the feasibility of DART ionization coupled to a high‐resolution mass spectrometer utilizing an orbitrap mass analyzer (orbitrap MS) for high‐throughput analysis of antiparasitic veterinary drugs was explored.

METHODS

To obtain the best DART‐orbitrap MS performance, stepwise optimization of instrumental parameter settings, such as ionization gas temperature and mass resolving power, was performed. The optimized method was applied to feed and bovine milk samples previously extracted following a QuEChERS‐like strategy.

RESULTS

Most antiparasitic drugs could be analyzed following the described method. Positive DART ionization provided the protonated molecules [M+H]+; in negative DART ion mode, deprotonated molecules [M–H]– were observed. As an exception, polyether ionophores could be observed as the sodiated adducts [M+Na]+. Samples of milk and feed were extracted using a modified QuEChERS method for the determination of benzimidazoles and coccidiostats respectively and quantification was carried out by matrix‐matched calibration curves.

CONCLUSIONS

The combination of an analysis time of less than 1 min per sample and the possibility to acquire accurate masses under high mass resolving power (HR) makes the DART‐HRMS technique an effective tool for rapid qualitative screening of antiparasitic veterinary drugs. Additionally, the results obtained in this study demonstrated the feasibility of this approach to quantify target analytes at levels down to 1 µg kg–1 for benzimidazolic compounds in milk and 0.25 mg kg–1 for coccidiostats in chicken feed. Copyright © 2012 John Wiley & Sons, Ltd.
Influence of matrix effect on the performance of the method for the official residue control of non‐steroidal anti‐inflammatory drugs in animal muscle
Authors: Olejnik Małgorzata , Jedziniak Piotr , Szprengier‐Juszkiewicz Teresa , Żmudzki Jan
Published: December 2012

RATIONALE

During the development and validation of mass spectrometry based method in residue control analysis, it is recommended to evaluate the level of the matrix effect. Often its level is relatively high, despite extensive sample purification.

METHODS

The matrix effect in the method for the determination of non‐steroidal anti‐inflammatory drugs in animal muscle was tested using a post‐extraction addition technique. The experiment was performed to assess the impact of chromatographic conditions and design of ion source on the results. Additionally, the impact of phospholipids was tested.

RESULTS

The matrix effect signal varied from 36% (64% ion suppression) to 192% (92% ion enhancement), depending on the analyte and species. The internal standard corrected matrix effect was generally lower but was still high for some analytes. Both chromatographic conditions and ion source design have influence on the level of matrix effect; however, this effect was no longer observed after compensation with internal standards.

CONCLUSIONS

Currently, no commonly accepted criteria exist for the interpretation of results of determination of matrix effects; such criteria have been proposed in this paper, based on guidelines for bioanalytical methods and results of the study. Copyright © 2012 John Wiley & Sons, Ltd.
Performance of dielectric barrier discharge ionization mass spectrometry for pesticide testing: a comparison with atmospheric pressure chemical ionization and electrospray ionization
Authors: Gilbert‐López Bienvenida , Geltenpoth Helma , Meyer Cordula , Michels Antje , Hayen Heiko , Molina‐Díaz Antonio , García‐Reyes Juan F. , Franzke Joachim
Published: December 2012

RATIONALE

The present study reports on the evaluation of dielectric barrier discharge microplasma ionization (DBDI) for liquid chromatography/high resolution mass spectrometry (LC/HRMS) analyses of pesticide residues in fruit and vegetables. Ionization, fragmentation, analytical performance and matrix effects displayed by LC/DBDI‐MS were critically evaluated and compared with both atmospheric pressure chemical ionization (APCI) and electrospray (ESI), using a set of over 40 representative multiclass pesticides.

METHODS

Sample preparation was accomplished using standard QuEChERS procedure and the identification and quantitation of the pesticides tested accomplished by means of LC/MS with a hybrid linear quadrupole ion trap (LIT)‐Fourier transform ion cyclotron resonance (FTICR) mass spectrometer operated in full‐scan positive ion mode using DBDI, APCI and ESI sources.

RESULTS

The developed LC/DBDI‐MS method allowed the screening of 43 pesticides in three different vegetable matrices: apple, orange and tomato. Minor matrix effects (i.e. signal suppression or enhancement ≤20%) were observed in most of the studied compounds: 95%, 70% and 81% of the studied compounds showed minor matrix effects in extracts of apple, orange and tomato, respectively. The results of the analysis of spiked orange extracts showed that the sensitivity obtained with LC/DBDI‐MS is appropriate for multi‐residue analysis of pesticide residues in fruit and vegetable samples. The limits of quantitation (LOQs) obtained for most of the studied pesticides were in compliance with the European Regulation 396/2005 (and subsequent updates) on food commodities (default maximum residue level of 10 µg kg–1).

CONCLUSIONS

Comparative studies with commercial sources demonstrate the suitability of DBDI as an ionization technique for residue analysis, because of the combination of the following two advantages: (1) the use of DBDI provides minimized matrix effects compared with APCI, and (2) improved the detection – in terms of sensitivity – of selected compounds that are not easily ionized by ESI, such as parathion. Copyright © 2012 John Wiley & Sons, Ltd.
Surface‐activated chemical ionization–electrospray mass spectrometry in the analysis of urinary thiodiglycolic acid
Authors: Puccio Giovanni , Brambilla Paolo , Conti Matteo , Bartolini Desirée , Noonan Douglas , Albini Adriana
Published: December 2012

RATIONALE

Thiodiglycolic acid (TDGA) is a urinary metabolite of the oxazaphosphorine class of chemotherapeutics, in particular of ifosfamide. Ifosfamide metabolism generates chloroacetaldehyde (CAA), a toxic compound associated with neurotoxicity, nephrotoxicity, urotoxicity and cardiotoxicity. CAA, in turn, interacts with cellular thiol groups leading to GSH depletion, cell death and generation of thiodiglycolic acid (TDGA), as a final product. TDGA is mainly excreted in the urine. The ability to accurately measure TDGA in urine, therefore, will be a useful way of monitoring exposure to ifosfamide during chemotherapy.

METHODS

TDGA in urine samples was measured with liquid chromatography coupled to mass spectrometry (LC/MS) by means of a novel Surface‐Activated Chemical Ionization–Electrospray (SACI–ESI) or a classical ESI ion source alone.

RESULTS

The SACI–ESI and ESI alone based methods for analysis of urinary TDGA were optimized and compared. A strong reduction in matrix effect together with enhanced quantification performances was obtained with the SACI–ESI when compared with ESI. In particular, an increase in quantification precision (from 85 to 95%) and accuracy (from 59 to 90%) were observed, which allowed for optimal detection of TDGA.

CONCLUSIONS

The LC/SACI–ESI‐MS approach provides a very sensitive and quantitative method for the analysis of TDGA. Thanks to the enhancement in sensitivity and matrix effect reduction, the SACI–ESI source enables the use of a relatively low‐cost ion‐trap mass spectrometer in the analysis of this toxicity biomarker in urine. Due to these characteristics, this approach would constitute an invaluable tool in the clinical laboratory, for measuring TDGA and other toxicity related biomarkers of chemotherapy with proper sensitivity and accuracy. Copyright © 2012 John Wiley & Sons, Ltd.