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.     

Quantification of genetically modified soya using strong anion exchange chromatography and time-of-flight mass spectrometry

Stable-isotope dimethyl labeling was applied to the quantification of genetically modified (GM) soya. The herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) was labeled using a dimethyl labeling reagent, formaldehyde-H₂ or -D₂. The identification and quantification of CP4 EPSPS was performed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The CP4 EPSPS protein was separated from high abundance proteins using strong anion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the tryptic peptides from the samples and reference were labeled with formaldehyde-H₂ and formaldehyde-D₂, respectively. The two labeled pools were mixed and analyzed using MALDI-MS. The data showed a good correlation between the peak ratio of the H- and D-labeled peptides and the GM soya percentages at 0.5, 1, 3, and 5 %, with R ² of 0.99. The labeling reagents are readily available. The labeling experiments and the detection procedures are simple. The approach is useful for the quantification of GM soya at a level as low as 0.5 %.     

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.     

Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrate the feasibility of using selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG), have good linear ranges, generally 10(3) to 10(4) . There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R(2) is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins within the range of 0.2-2 fmol, which is comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R(2)=0.928 and 0.851, respectively). However, the correlation between SRM/MRM and ELISA for CP is less desirable (R(2)=0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides being analyzed (R(2)=0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). The SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or LUMINEX are not available.     

A method combining SPITC and 18O labeling for simultaneous protein identification and relative quantification

The relative quantification and identification of proteins by matrix‐assisted laser desorption ionization time‐of‐flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N‐terminal derivatization with 4‐sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while ¹⁸O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4‐sulphophenyl isothiocyanate derivatization with ¹⁸O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R² value = 0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. Copyright © 2014 John Wiley & Sons, Ltd.     

Isotope coded protein label quantification of serum proteins—Comparison with the label-free LC-MS and validation using the MRM approach

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance.     

MALDI imaging directed laser ablation tissue microsampling for data independent acquisition proteomics

A multimodal workflow for mass spectrometry imaging was developed that combines MALDI imaging with protein identification and quantification by liquid chromatography tandem mass spectrometry (LC‐MS/MS). Thin tissue sections were analyzed by MALDI imaging, and the regions of interest (ROI) were identified using a smoothing and edge detection procedure. A midinfrared laser at 3‐μm wavelength was used to remove the ROI from the brain tissue section after MALDI mass spectrometry imaging (MALDI MSI). The captured material was processed using a single‐pot solid‐phase‐enhanced sample preparation (SP3) method and analyzed by LC‐MS/MS using ion mobility (IM) enhanced data independent acquisition (DIA) to identify and quantify proteins; more than 600 proteins were identified. Using a modified database that included isoform and the post‐translational modifications chain, loss of the initial methionine, and acetylation, 14 MALDI MSI peaks were identified. Comparison of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the identified proteins was achieved through an evolutionary relationships classification system.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Development of a high‐performance liquid chromatography method with fluorescence detection for the routine quantification of tamoxifen,endoxifen and 4‐hydroxytamoxifen in plasma from breast cancer patients

To date, several methods for the quantification of tamoxifen and its metabolites have been developed, most of which employ liquid chromatography tandem–mass spectrometry (LC–MS/MS). These methods are highly sensitive and reproducible, but are also time‐consuming and require expensive equipment; one of their main disadvantages is matrix ionization effects. A more viable option, particularly in developing countries, is high‐performance liquid chromatography coupled with UV or fluorescence detection. We developed and validated a method for simultaneous quantification of tamoxifen, endoxifen and 4‐hydroxytamoxifen based on high‐performance liquid chromatography with fluorescence detection in a reverse‐phase column. The method is rapid (16 min plus 5 min of column re‐equilibrium), accurate (80–100%) and precise (0.23–6.00%), and does not require any additional irradiation process. Sample pretreatment consists of protein precipitation with acetonitrile under alkaline conditions, employing only 200 μL plasma. The validated method's wide range allowed quantification of steady‐state levels in patients under standard tamoxifen treatment (20 mg/day). This assay is ready for application in clinical studies and routine quantification of tamoxifen, endoxifen and 4‐hydroxytamoxifen in healthcare institutions.     

Quantification of intact covalently metal labeled proteins using ESI‐MS/MS

Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide‐ion‐based tags from the quantification using inductively coupled plasma‐MS into the quantification of labeled intact proteins using electrospray ionization (ESI)‐MS and ESI‐MS/MS. We applied the metal chelate tag MeCAT‐iodoacetamide (IA) (1,4,7,10‐tetraazacyclododecane N,N′,N″,N″ ′‐tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3‐reversed phase‐high‐performance liquid chromatography interfaced to ESI‐MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT‐IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance‐MS or higher‐energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high‐performance liquid chromatography/ESI‐MS/MS quantification data were validated using inductively coupled plasma‐MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent. Copyright © 2014 John Wiley & Sons, Ltd.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

A Sandwich‐type Electrochemiluminescence Aptasensor for Thrombin Based on Functional Co‐polymer Electrode Using Ru(bpy)32+ Doped Nanocomposites as Signal‐amplifying Tags

Herein, a signal‐on sandwich‐type electrochemiluminescence (ECL) aptasensor for the detection of thrombin (TB) was proposed. The graphene (GR) doped thionine (TH) was electropolymerized synchronously on the bare glassy carbon electrode (GCE) to form co‐polymer (PTG) electrode. The gold nanoparticles (AuNPs) were decorated on the surface of the PTG by in‐situ electrodeposition, and the functional co‐polymer (PTG‐AuNPs) electrode was utilized as sensing interface. Then, TB binding aptamer I (TBA I) as capture probes were modified on the PTG‐AuNPs electrode to capture TB, and Ru(bpy)₃²⁺/silver nanoparticles doped silica core‐shell nanocomposites‐labeled TB binding aptamer II (RuAg/SiO₂NPs@TBA II) were used as signal probes to further bind TB, resulting in a sandwich structure. With the assistant of silica shell and AgNPs, the enrichment and luminous efficiency of Ru(bpy)₃²⁺ were significantly improved. Under the synergy of PTG‐AuNPs and RuAg/SiO₂NPs, the ECL signal was dramatically increased. The proposed ECL aptasensor displayed a wide linear range from 2 fM to 2 pM with the detection limit of 1 fM, which is comparable or better than that in reported ECL aptasensors for TB using Ru(bpy)₃²⁺ and its derivatives as the luminescent substance. The excellent sensitivity makes the proposed aptasensor a promising potential in pharmaceutical and clinical analysis.     

Evaluation of peptide fractionation strategies used in proteome analysis

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2‐D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong‐cation exchange, hydrophilic interaction chromatography, alkaline‐RP and solution isoelectric focusing, which can be used prior to LC‐MS/MS analysis, were compared. Strong‐cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide‐based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.     

A Sensitive and Selective Fluorescent Probe for Cysteine Based on a New Response‐Assisted Electrostatic Attraction Strategy: The Role of Spatial Charge Configuration

A new strategy for fast fluorescent detection of cysteine (Cys), based on a response‐assisted electrostatic attraction, is demonstrated. By utilizing this strategy, we designed and synthesized three fluorescent probes for the specific detection of Cys under actual physiological conditions. The probe m‐ CP , a coumarin fluorophore conjugated with a substituted methyl pyridinium group through an unsaturated ketone unit, showed highly selective and sensitive detection for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The kinetic analysis indicated that the sensing process was highly accelerated (a response time less than 1 min) by the response‐assisted electrostatic attraction. More importantly, control experiments with isomeric probes first demonstrated that the spatial charge configuration of the probe played an important role in Cys‐preferred selectivity and kinetic rate acceleration. Furthermore, the practical utility of the probe m‐ CP in the fluorescent labeling of Cys residues within proteins was demonstrated. Finally, these probes were employed in living cell imaging with HeLa cells, in which it displayed satisfactory cell permeability and enabled us to distinguish active thiols in the cytoplasm, nucleus, and mitochondria.     

Gold Nanoparticles as Dual Functional Sensor to Detect E.coliDH5α as a Model for Gram‐negative Bacteria

4‐aminothiophenol‐modified gold nanoparticles (PATP‐AuNPs) were used as colorimetric and Surface Enhanced Raman Scattering (SERS) probes for the sensitive detection of Escherichia coliDH5α, as a model for Gram‐negative bacteria. The nano‐probes were easy to prepare through Au‐S bonding. Under optimized conditions, the PATP‐AuNPs surface with positive charge can bind with negatively charged E.coliDH5α via electrostatic adhesion, resulting in a quick color change from red to blue, and also a dramatic SERS signal enhancement from thousands of AuNPs aggregated on the surface of bacteria, which was utilized for both colorimetric and SERS detection of E.coliDH5α. For colorimetric analysis, it is the first time that the classical partial least square (PLS) regression was utilized to deal with the relationship between adsorption and E.coliDH5α concentrations. Excellent linear relationship was observed from 1.1 x 10⁷ to 1.3 x 10⁸ cfu mL^‐1 with the average relative error (ARE) of 5.430, which was more accurate than the traditional extinction ration method. When coupled with confocal Raman microscope, this PATP‐AuNPs probes could be used to detection SERS signals produced from even one single bacterium. This bioassay is rapid, less expensive and convenient for bacteria detection and analysis. Therefore, PATP‐AuNPs system as a novel, versatile, on‐site and real‐time Gram‐negative bacteria sensor, would have a wide range of practical applications.     

Rapid and sensitive identification of ricin in environmental samples based on lactamyl agarose beads using LC‐MS/MS (MRM)

Ricin, a plant‐derived toxin extracted from the seeds of Ricinus communis (castor bean plant), is one of the most toxic proteins known. Ricin's high toxicity, widespread availability, and ease of its extraction make it a potential agent for bioterrorist attacks. Most ricin detection methods are based on immunoassays. These methods may suffer from low efficiency in matrices containing interfering substances, or from false positive results due to antibody cross reactivity, with highly homologous proteins. In this study, we have developed a simple, rapid, sensitive, and selective mass spectrometry assay, for the identification of ricin in complex environmental samples. This assay involves three main stages: (a) Ricin affinity capture by commercial lactamyl‐agarose (LA) beads. (b) Tryptic digestion. (c) LC‐MS/MS (MRM) analysis of tryptic fragments. The assay was validated using 60 diverse environmental samples such as soil, asphalt, and vegetation, taken from various geographic regions. The assay's selectivity was established in the presence of high concentrations of competing lectin interferences. Based on our findings, we have defined strict criteria for unambiguous identification of ricin. Our novel method, which combines affinity capture beads followed by MRM‐based analysis, enabled the identification of 1 ppb ricin spiked into complex environmental matrices. This methodology has the potential to be extended for the identification of ricin in body fluids from individuals exposed (deliberately or accidentally) to the toxin, contaminated food or for the detection of the entire family of RIP‐II toxins, by applying multiplex format.     

Fast quantification of cyclophosfamide and its N‐dechloroethylated metabolite 2‐dechloroethylcyclophosphamide in human plasma by UHPLC‐MS/MS

A rapid, novel and reliable UHPLC‐MS/MS method was developed and validated for simultaneous determination of cyclophosphamide (CP) and its dechloroethylated metabolite, 2‐dechloroethylcyclosphamide (2‐DCECP) in human plasma. The plasma samples were conducted by protein precipitation with 3‐fold acetonitrile, containing 0.1% formic acid. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode, using tinidazole as internal standard (IS). Chromatographic separation was performed on an Agilent poroshell 120 SB‐C₁₈ column (2.1 × 75 mm, 2.7 µm) using gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.5 mL/min, the total run time was 2.5 min. The limit of quantification (LOQ) was 20 ng/mL for both CP and 2‐DCECP. Accuracies and precisions were <15% at LOQ and below 10% at quality control concentration levels. This UHPLC‐MS/MS method was successfully applied for the estimation of CP and 2‐DCECP in human plasma, which was also useful for clinical toxicology studies and therapeutic drug monitoring of CP. Copyright © 2014 John Wiley & Sons, Ltd.     

New insights on proteomics of transgenic soybean seeds: evaluation of differential expressions of enzymes and proteins

This work reports the evaluation of differentially expressed enzymes and proteins from transgenic and nontransgenic soybean seeds. Analysis of malondialdehyde, ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), and catalase (EC 1.11.1.6) revealed higher levels (29.8, 30.6, 71.4, and 35.3%, respectively) in transgenic seeds than in nontransgenic seeds. Separation of soybean seed proteins was done by two-dimensional polyacrylamide gel electrophoresis, and 192 proteins were identified by matrix-assisted laser desorption/ionization (MALDI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) and electrospray ionization (ESI) QTOF MS. Additionally, the enzyme CP4 EPSPS, involved in the genetic modification, was identified by enzymatic digestions using either trypsin or chymotrypsin and ESI-QTOF MS/MS for identification. From the proteins identified, actin fragment, cytosolic glutamine synthetase, glycinin subunit G1, and glycine-rich RNA-binding protein were shown to be differentially expressed after analysis using the two-dimensional difference gel electrophoresis technique, and applying a regulator factor of 1.5 or greater.     

Solid‐phase microextraction with fast GC combined with a high‐speed triple quadrupole mass spectrometer for targeted and untargeted food analysis

The present contribution is focused on the evaluation of a high‐speed triple quadrupole mass spectrometer, carried out under moderately fast GC conditions (analysis time: 16.6 min). The mass spectrometric instrument can be operated under high‐speed GC conditions, in both full‐scan (maximum scan speed: 20 000 amu/s) and multiple reaction monitoring (MRM) modes (minimum dwell time: 0.01 s). Additionally, the mass spectrometric system can generate full scan and MRM information, simultaneously and rapidly. A headspace solid‐phase microextraction with fast GC coupled to triple quadrupole MS approach was developed for the: (i) qualitative untargeted analysis of brewed tea volatiles, and (ii) MRM qualitative and quantitative analysis of targeted volatiles (also in brewed tea), namely 30 phytosanitary contaminants. The performance of the triple quadrupole instrument was satisfactory both for identification and quantification purposes. Furthermore, the method sensitivity was more than sufficient for the requirements of current legislation. Method validation, related to the MRM analysis, was performed considering: precision of quantification data (maximum coefficient of variation value: 12.0%) and quantification/qualification ion ratios (maximum coefficient of variation value: 14.4%), along with limits of detection (4 parts per trillion–5 parts per billion range) and quantification (14 parts per trillion–16 parts per billion range).     

Real-time detection of genetically modified soya using Lightcycler and ABI 7700 platforms with TaqMan,Scorpion, and SYBR Green I chemistries

A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.