Mass spectrometric detection of CP4 EPSPS in genetically modified soya and maize

The potential of protein fractionation hyphenated to mass spectrometry (MS) to detect and characterize the transgenic protein present in Roundup Ready soya and maize has been investigated. Genetically modified (GM) soya and maize contain the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Agrobacterium tumefaciens CP4, which confers resistance to the herbicide glyphosate. The GM soya and maize proteomes were fractionated by gel filtration, anion-exchange chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) prior to MS. This facilitated detection of a tryptic peptide map of CP4 EPSPS by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and nanoelectrospray ionization quadrupole time-of-flight (nanoESI-QTOF) MS. Subsequently, sequence information from the CP4 EPSPS tryptic peptides was obtained by nanoESI-QTOF MS/MS. The identification was accomplished in 0.9% GM soya seeds, which is the current EU threshold for food-labeling requirements.     

Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya

The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study.     

Mass spectrometry in the structural analysis of flavonoids

Flavonoids are very common and widespread secondary plant metabolites. They have a wide range of biological and physiological activities and serve as chemotaxonomic marker compounds. Therefore, they have been extensively investigated both in the past and during recent years. The interest in them is still increasing. In the search for new compounds, and also in quality control, there is a need to have reliable methodology for the analysis of flavonoids. Mass spectrometry can make an invaluable contribution because of its high sensitivity, possibilities of coupling with liquid chromatography and the availability of powerful tandem mass spectrometric techniques. A review of currently available mass spectrometric methodology used in the structure elucidation of flavonoids is presented. Sample preparation, liquid chromatographic/mass spectrometric analysis and tandem mass spectrometric procedures for the characterization of flavonoid aglycones, O-glycosides, C-glycosides and acylated glycosides are considered.     

Mass probe-assisted ionization method for total analysis of biomolecules with electrospray ionization-mass spectrometry

In this paper, we review the mass probes used for the derivation of a variety of biomolecules efficiently detected by the electrospray ionization-mass spectrometry and mass probe-assisted ionization method for total analysis and determination by consecutive detection with a single instrument. We describe mass probes for a variety of molecules including proteins, nucleobases, metallic cations, and other small molecules.     

Affinity-based mass spectrometry using magnetic iron oxide particles as the matrix and concentrating probes for SALDI MS analysis of peptides and proteins

Silane-immobilized magnetic iron oxide particles were used as the assisting material in surface-assisted laser desorption/ionization (SALDI) mass spectrometric analysis. This approach can be used to analyze small proteins and peptides. The upper detectable mass range is approximately 16 kDa. The detection limit for peptides is about 20 fmol. Silanized iron oxide particles with negatively charged functionalities can also be used as the affinity probes to selectively trap oppositely charged species from sample solutions by adjusting the pH of the solution. A tryptic digest product of cytochrome C at a concentration as low as 10 nM can be enriched by the particles and directly analyzed by iron oxide SALDI MS without the need for elution steps. Affinity-based mass spectrometry using the bifunctional silanized magnetic iron oxide particles as the SALDI matrix and concentrating probe is demonstrated in this study.     

Mass spectrometry and partial least-squares regression: a tool for identification of wheat variety and end-use quality

Rapid methods for the identification of wheat varieties and their end-use quality have been developed. The methods combine the analysis of wheat protein extracts by mass spectrometry with partial least-squares regression in order to predict the variety or end-use quality of unknown wheat samples. The whole process takes approximately 30 min. Extracts of alcohol-soluble storage proteins (gliadins) from wheat were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Partial least-squares regression was subsequently applied using these mass spectra for making models that could predict the wheat variety or end-use quality. Previously, an artificial neural network was used to identify wheat varieties based on their protein mass spectra profiles. The present study showed that partial least-squares regression is at least as useful as neural networks for this identification. Furthermore, it was demonstrated that partial least-squares regression could be used to predict wheat end-use quality, which has not been possible using neural networks.     

Facile synthesis of Fe3O4@polyethyleneimine modified with 4‐formylphenylboronic acid for the highly selective extraction of major catecholamines from human urine

The levels of catecholamines, especially dopamine, epinephrine and norepinephrine in urine and plasma have been used to assist the diagnosis and treatment of psychosis. Due to their low endogenous concentrations, the determination of the three major catecholamines is very difficult. Boronate adsorbents are often employed to extract these cis‐diol compounds from complex matrices. In this work, a novel type of magnetic nanoparticles modified with 4‐formylphenylboronic named Fe₃O₄@PEI‐FPBA was synthesized by a facile two‐step approach. The abundant amino groups of polyethyleneimine provided the rich binding sites for boronate ligands. Herein, the adsorption capacity of Fe₃O₄@PEI‐FPBA is greatly improved with a value of 3.45 mg/g towards epinephrine, which is much larger than that of analogous material without polyethyleneimine. The magnetic nanoparticles also exhibited high magnetization (72.25 emu/g) and specific selectivity towards the catecholamines. Finally, a liquid chromatography tandem mass spectrometry method based on Fe₃O₄@PEI‐FPBA nanoparticles was successfully used to determine the three catecholamines from human urine samples. The linearity, limit of quantitation, recovery and precision of the method were satisfactory. Based on the method, it is found that the levels of dopamine, epinephrine and norepinephrine in depressive patients are higher than those in healthy controls.     

MALDI‐TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varieties

A procedure for identification of malting barley varieties using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of ethanol‐soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI‐TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry‐based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd.     

新型包覆Pd纳米粒子液相色谱-串联质谱法用于鉴定沙特阿拉伯原油中的含氧化合物(英文)

Saudi Arabian crude oil is a super complex mixture and,up to now,there has been little research into its heteroatom-containing compounds.First,oxygenated compounds(OCs)were isolated from Saudi Arabian oil using a Pd nanoparticle exchange complex,which formed between the nano-Pds and the oxygenated ligands.Normally,polycyclic aromatic sulphur heterocycles(S-PAHs)are separated from petroleum oil via the same method.The obtained results reveal that all the OC formulations with S-PAHs can be separated from the pre-isolated aromatic fraction of crude oil via this approach.S-PAHs are mixtures of benzothiophene and dibenzothiophene congeners.The isolated OCs are composed mainly of hydroxyl compounds.The liquid chromatography(LC)/electrospray ionization(ESI)in positive ion mode ESI(+)/tandem mass spectrometry(MS/MS)technique was used to assign the molecular weight distribution and identify the isolated OCs.The LC/ESI(+)-MS/MS technique differentiates S-PAHs and OCs using protonated ions.Thus,LC/ESI(+)-MS/MS can be used to assign molecular weight distributions for both the groups as a single mixture.MS/MS in precursor ion mode was used for the immediate identification of the target S or O analytes.     

改良的QuEChERS结合气相色谱-质谱法测定新鲜水果和肉中肉桂醛残留

建立了改良的QuEChERS技术结合气相色谱-质谱检测水果和肉中肉桂醛残留量的定性定量分析方法。试样用乙酸乙酯溶液均质提取,经N-丙基乙二胺（PSA）、石墨化炭黑（GCB）、C₁₈固相吸附剂净化,在电子轰击电离（EI）源、选择离子监测（SIM）模式下检测,外标法定量。肉桂醛在0.01~0.5 mg/L范围内线性关系良好,相关系数大于0.999。水果和鲜肉中肉桂醛的定量限分别为0.5 mg/kg和1.0 mg/kg。在3个添加水平下,肉桂醛的加标回收率为81.9%~104.5%,相对标准偏差（RSD,n=6）为1.1%~8.6%。该方法操作简单,净化效果好,适用于水果和鲜肉中肉桂醛残留量的快速检测。     

蛋白质组学中质谱分析前的预富集研究进展

回顾了近年来生物质谱分析鉴定蛋白质及多肽前的预富集方法研究进展,其中包括固相微萃取富集方法、靶上富集方法、电洗脱富集方法以及磷酸化蛋白质/肽段的富集方法,总结和归纳了各种方法所具有的优缺点,引用文献59篇。     

Immunoaffinity chromatographic and immunoprecipitation methods combined with mass spectrometry for characterization of circulating transthyretin

Transthyretin (TTR) is a small serum protein that is involved in distinct phenotypes of amyloidosis with different tissue localization, age of onset, and rate of progression. Some types of TTR-amyloidosis (such as familial amyloid polyneuropathy) are associated with various amino acid point mutations, while cardiac amyloid myopathy may also be associated with precipitation of the wild-type molecules, e.g., in senile systemic amyloidosis. Because of the unsettled relationship between circulating and precipitated TTR we here explore on-line immunoaffinity (IA) chromatography-MS and immunoprecipitation (IP)-MS methods for characterizing the circulating TTR population in normal individuals and in patients with known TTR-amyloidosis. It was found necessary to reduce the samples, e.g., with DTT, prior to ESI-TOF-MS. This reversed oxidative modifications to sufficiently resolve the two mass peaks isolated from sera of heterozygous patients. A simple IP technique without the use of centrifugal filtration was found to be convenient for the assessment of the TTR population in serum as demonstrated for both normal and variant (the Met111Leu mutation) TTR. This approach also readily allowed the identification of oxidation, S-sulfation, and S-cysteinylation in unreduced samples, while these modifications were less well resolved in the on-line IA chromatography-MS approach.     

Proteotypic peptides of hairs for the identification of common European domestic and wild animal species revealed by in-sample protein digestion and mass spectrometry analysis

The aim of this work is to offer an alternative or complementary analytical tool to the time-consuming and expensive methods commonly used for the recognition of animal species according to their hair. The paper introduces a simple and fast way for species differentiation of animal hairs called in-sample digestion. A total of 10 European animal species, including cat, cow, common degu, dog, fallow deer, goat, horse, sika deer, rabbit, roe deer, and 17 different breeds of dogs were examined using specific tryptic cleavage directly in hair followed by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-electrospray ionization quadrupole time of flight. Principal component analysis was used for the subsequent mass spectrometric data evaluation. This novel approach demonstrates the ability to distinguish among individual animal species, which is supported by finding characteristic m/z values obtained by the mass spectrometry for each animal species. The approach was successfully tested on two “blind” samples. On the other hand, the attempt to distinguish among hairs of different dog breeds has not been successful due to the very similar protein composition and their amino acid sequences.     

质谱技术在缺血性中风标志物发现中的应用及进展

脑卒中是世界上主要的致死和致残的原因之一,缺血性脑卒中(IS)约占所有脑卒中案例的87%。由于IS存在黄金3或4.5 h的治疗窗,其早期筛查和快速诊断显得尤为重要。分子标志物的发现为IS的预测和诊断提供了新的思路。质谱技术可以快速灵敏地检测生物医学样本中的各种化合物,已被广泛应用于IS生物标志物的发现。该综述总结和讨论了质谱在IS标志物(特别是蛋白和小分子代谢物标志物)发现中的应用及其最新进展。     

Analysis of amicarbazone and its two metabolites in grains and soybeans by liquid chromatography with tandem mass spectrometry

A sensitive, simple and reliable analytical method based on a modified quick, easy, cheap, effective, rugged, safe sample preparation and liquid chromatography with tandem mass spectrometry detection was developed for the simultaneous determination of amicarbazone and its two major metabolites desamino amicarbazone and isopropyl‐2‐hydroxy‐desamino amicarbazone residues in grains (rice, wheat, corn, buckwheat) and soybean. Several parameters, including liquid chromatography and tandem mass spectrometry conditions, extraction approaches and the adsorbents for clean‐up, which might influence the accuracy of the method, were extensively investigated. The established method was further validated by determining the linearity (R² > 0.99), fortified recovery (79–118%), precision (1–12%) and sensitivity (limit of quantification, 5 μg/kg for amicarbazone and desamino amicarbazone, and 10 μg/kg for isopropyl‐2‐hydroxy‐desamino amicarbazone). Finally, the established method was successfully applied to determine the residues of amicarbazone and its metabolites in 49 real samples of grain and soybean.     

聚苯胺修饰电极-电化学氢化物发生原子荧光光谱法测定食品中锡含量

运用以聚苯胺修饰的石墨电极为阴极的电化学氢化物发生装置实现了锡元素的电化学氢化物发生.在电极表面聚合一层聚苯胺,能够有效地提高锡元素的电化学氢化物发生效率,通过与原子荧光光谱仪联用,成功地测定了食品中的锡含量.本工作对各种实验参数和干扰情况进行了详细研究.方法对锡的检出限为1.2 ng/mL(3σ);样品分析精密度(R...     

Suitability of selective pressurized liquid extraction combined with gas chromatography-ion-trap tandem mass spectrometry for the analysis of polybrominated diphenyl ethers

A one-step extraction and clean-up method using pressurized liquid extraction (PLE) (selective PLE) combined with gas chromatography-ion-trap tandem mass spectrometry (GC-ITMS-MS) was evaluated for the analysis of polybrominated diphenyl ethers (from tri- to hepta-PBDEs) at low concentrations in fish and shellfish samples. To this end, the performance of an on-line PLE extraction/clean-up method and of a classical Soxhlet extraction and clean-up method using a multi-layer modified silica column were compared. The two sample treatment methods provided similar results, although an important reduction in the sample treatment time (40 min per sample) was achieved using the selective PLE method. In addition, the suitability of the PLE combined with GC-ITMS-MS method was evaluated by comparing the results obtained in the analysis of fish samples with those obtained by gas chromatography-high resolution mass spectrometry (GC-HRMS). Good agreement between both techniques was obtained with differences between the mean values of less than 16%. The selective PLE method coupled to GC-ITMS-MS produced accurate results for PBDE determination with low limits of detection (1.0-16.8 pg g⁻¹ wet weight) and quantification (3.1-51 pg g⁻¹ wet weight) as well as good precision (RSD < 16%). This method has been applied to the analysis of PBDEs in fish and shellfish samples collected at fish markets in Catalonia (NE Spain).     

Mass distribution for the microextraction of polycyclic aromatic hydrocarbon metabolites investigated with fluorescence spectrometry

Mass-balance data were acquired using fluorescence spectrometry for 2-naphthol and three polycyclic aromatic hydrocarbon (PAH) metabolites using liquid-liquid-liquid microextraction and liquid-liquid microextraction systems. The PAH metabolites are very important biomarkers, and there has been no previously reported mass-balance data on these compounds with microextraction systems. In addition, the effects of two solvent systems used in the preparation of donor and acceptor phases were investigated. The effects of the solvent systems on the partitioning process were compared. The mass balance results showed that significant amounts of the hydrophobic metabolites were held in the pores of the microfiber in both the three-phase and two-phase microextraction systems. Based on the mass-balance data, a new enrichment factor was defined as the concentration of the solute trapped in the pores of the fiber divided by the initial concentration of the solute in the donor phase. Because of the relatively large amount of the solute in the pores, it is envisioned that the solutes could be easily extracted from the pores, the extraction solvent concentrated, and further separated by capillary electrophoresis or characterized by solid-matrix phosphorescence, solution fluorescence, or mass spectrometry.