Direct Infusion Electrospray Mass Research Articles

Chemical Constituents and Cytotoxic Activity of Rhinella jimi (Anura: Bufonidae)

Rhinella jimi toads (Stevaux, 2002) belong to the Bufonidae family, are endemic in the Brazilian Northeast and are commonly found during rainy periods. In general, amphibians of this family have in their poisons different metabolites that show a diversity of pharmacological activities. The isolation and identification of these compounds are of great importance, and techniques such as high-performance liquid chromatography coupled to mass spectrometry are widely used for the discovery of novel and known compounds in these poisons. For R. jimi poison, the ethyl acetate and methanolic extracts were obtained and thirty compounds were identified by combining ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) with direct infusion atmospheric pressure chemical ionization mass spectrometry (DI-APCI-MS/MS) and direct infusion electrospray mass spectrometry (DI-ESI-MS/MS) for each extract, respectively. Marinobufagin (2) and marinobufotoxin (19) were the majorities of each extract, respectively. In addition, other bufadienolides mainly present in the ethyl acetate extract, such other bufotoxins, alkaloids and arginine diacid derivatives were identified in the methanol extract. In a cytotoxic assay by 3-(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide (MTT), the extracts and compound 2 demonstrated half-maximal inhibitory concentration (IC50) values better than the positive control doxorubicin, evidencing excellent cytotoxic. This is the most complete study of the chemical composition of R. jimi toad poison and its respective cytotoxic activity, promoting the enrichment of knowledge about this family and species.

The Inflammatory Lipid, 2‐Chlorohexadecanal: Cell Toxicity and Potential Scavenger Protection

Hypochlorous acid is produced by either leukocyte myeloperoxidase activity or from chlorine gas reactions with water. 2‐Chlorofatty aldehydes (2‐ClFALD) are: 1) formed when hypochlorous acid attacks the plasma membrane phospholipid plasmalogen molecular subclass and 2) produced following leukocyte activation as well in the lung of mice exposed to chlorine gas. The biological role of 2‐ClFALD is largely unknown. Recently, our lab showed the electrophilic α‐chlorinated carbon of the 2‐ClFALD molecular species, 2‐chlorohexadecanal (2‐ClHDA) is targeted by nucleophilic attack of the cysteinyl‐sulfhydryl group of glutathione. Here, we synthesized an alkyne analog of 2‐ClHDA (2‐ClHDyA) to demonstrate 2‐ClHDyA protein modification in RAW 264.7 cells using click chemistry strategies. Furthermore, confocal microscopy showed that 2‐ClHDyA co‐localizes to the endoplasmic reticulum, Golgi, and mitochondria. Additionally, diffuse distribution of 2‐ClHDyA suggested cytoplasmic protein modification. The intensity of the fluorescence signal of 2‐ClHDyA increased in a dose‐dependent manner suggesting increased protein modification. Cell viability/metabolic activity assessed by 3‐(4,5‐Dimethyl‐2‐Thiazolyl)‐2,5‐Diphenyl‐2H‐Tetrazolium Bromide (MTT) test revealed that 2‐ClHDA reduced RAW 264.7 cell viability in a dose‐dependent manner. Cell viability was further reduced in RAW 264.7 cells treated with 2‐ClHDA in DMEM with Fetal Bovine Serum (FBS) supplementation. Click chemistry showed 2‐ClHDyA modified FBS proteins. These observations suggested 2‐ClFALD modified proteins might have accounted for cell death via cell signaling outside or inside the cell. Next, we investigated traditional aldehyde sequesterers including N‐acetyl cysteine (NAC), carnosine, and aminoguanidine to identify potential protectors that would prevent cellular 2‐ClFALD‐protein interaction resulting in reduced toxicity caused by 2‐ClFALD. Among tested sequesterers, NAC proved to be an effective sequesterer of 2‐ClHDA. NAC prevented 2‐ClHDyA interaction with FBS proteins and rescued cell death significantly. Next, we characterized the conjugate of 2‐ClHDA and NAC using direct infusion electrospray mass spectrometry. The conjugate appeared to be a result of a nucleophilic substitution reaction where nucleophilic sulfur of NAC attacked the alpha carbon of 2‐ClHDA leading to the release of chloride ion. These studies demonstrate that the interaction of 2‐ClFALD with cellular proteins with free sulfhydryl groups cause toxicity and the interaction of 2‐ClFALD with N‐acetyl cysteine may protect against toxicity caused by 2‐ClFALD.Support or Funding InformationNIH grants: R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697 and U01 ES028182

Identification of the Side Products That Diminish the Yields of the Monoamidated Product in Metal-Catalyzed C-H Amidation of 2-Phenylpyridine with Arylisocyanates.

The Ru(II)-catalyzed amidation of 2-arylpyridines with aryl isocyanates via C-H bond activation is less efficient than described previously, due to the formation of a series of side products, which were readily identified using direct infusion electrospray mass spectrometry and high-performance liquid chromatography-mass spectrometry.

Direct infussion Electrospray Mass Spectrometry as a new non-invasive tool for serum metabolomics in induced-stress subjects

Background and Objectives: Nanotechnology is becoming a tool for the study of changes in the metabolome of patients in different states of disease. Analytical techniques such as Electrospray Mass Spectrometry, allow to find biomarkers by determi- nation of metabolites. Nowadays, there is not an objective analytical approach for diagno- sis of stress. Thus, the objectives of this pilot work are: - Describing the development of a fast, direct and non-invasive analytical protocol, ap- plied for the first time, to study the metabolomic profile of patient s different states through a disease. - Testing the protocol in a pilot sample with non-stressed and stress-induced subjects. Methods: High resolution direct infusion electrospray mass spectrometry has been used to analyse the metabolome of blood samples (0.3 ml) from six subject s. Results: Data prove a clear discrimination between non-stressed and stressed states in the metabolome. Data showed different predominant metabolites in both states. Results allow objective characterization of the state of the patient. Conclusions: Although this is a pilot study, the method was successful in discriminating different metabolites in non-stressed and stress-induced subjects.

Application of metabolomics based on direct mass spectrometry analysis for the elucidation of altered metabolic pathways in serum from the APP/PS1 transgenic model of Alzheimer's disease

Metabolomic analysis of brain tissue from transgenic mouse models of Alzheimer's disease has demonstrated a great potential for the study of pathological mechanisms and the development of new therapies and biomarkers for diagnosis. However, in order to translate these investigations to the clinical practice it is necessary to corroborate these findings in peripheral samples. To this end, this work considers the application of a novel metabolomic platform based on the combination of a two-steps extraction procedure with complementary analysis by direct infusion electrospray mass spectrometry and flow infusion atmospheric pressure photoionization mass spectrometry for a holistic investigation of metabolic abnormalities in serum samples from APP/PS1 mice. A number of metabolites were found to be perturbed in this mouse model, including increased levels of di- and tri-acylglycerols, eicosanoids, inosine, choline and glycerophosphoethanolamine; reduced content of cholesteryl esters, free fatty acids, lysophosphocholines, amino acids, energy-related metabolites, phosphoethanolamine and urea, as well as abnormal distribution of phosphocholines depending on the fatty acid linked to the molecular moiety. This allowed the elucidation of possible pathways disturbed underlying to disease (abnormal homeostasis of phospholipids leading to membrane breakdown, energy-related failures, hyperammonemia and hyperlipidemia, among others), thus demonstrating the utility of peripheral samples to investigate pathology in the APP/PS1 model.

Styrene polymerization efficiently catalyzed by iron-containing imidazolium-based ionic liquids: Reaction mechanism and enhanced ionic liquid effect

Iron-containing imidazolium-based ionic liquids were successfully applied as catalysts in the styrene polymerization. Solventless conditions, organic solvents and different imidazolium-based ionic liquid catalysts were tested to evaluate the best reaction conditions. The ionic liquids BMI.FeCl4 and BMI.Fe2Cl7 showed very distinct catalytic activities. Schulz–Flory distributions indicate two or three active catalytic species in different distributions. Via on-line direct infusion electrospray (tandem) mass spectrometry (ESI-MS(/MS)) reaction monitoring, both active catalytic species in accordance with the Schulz–Flory distributions were intercepted and characterized, providing important mechanistic insights that allowed us to propose a rationale for the origin of the superior catalytic efficiency.

Development of a metabolomic approach based on urine samples and direct infusion mass spectrometry

The analysis of urine by direct infusion mass spectrometry suffers from ion suppression due to its high salt content and inter-sample variability caused by the differences in urine volume between persons. Thus, urine metabolomics requires a careful selection of the sample preparation procedure and a normalization strategy to deal with these problems. Several approaches were tested for metabolomic analysis of urine samples by direct infusion electrospray mass spectrometry (DI–ESI–MS), including solid phase extraction, liquid–liquid extraction, and sample dilution. In addition, normalization of results based on conductivity values and statistical treatment was performed to minimize sample variability. Both urine dilution and solid phase extraction with mixed mode sorbent considerably reduced the salt content in urine, providing comprehensive metabolomic fingerprints. Moreover, statistical data normalization enabled the correction of inter-sample physiological variability, improving the quality of results obtained. Therefore, high-throughput DI–ESI–MS fingerprinting of urine samples can be achieved with simple pretreatment procedures allowing the use of this noninvasive sampling in metabolomics. Finally, the optimized approach was tested in a pilot metabolomic investigation of urine samples from transgenic mice models of Alzheimer’s disease (APP/PS1) in order to illustrate the potential of the methodology.

Human serum albumin cysteinylation is increased in end stage renal disease patients and reduced by hemodialysis: mass spectrometry studies

The aim of the present work was to monitor the covalent modifications of human serum albumin (HSA) in end stage renal diseases (ESRD) non-diabetic patients, before and after hemodialysis (HD), by direct infusion electrospray mass spectrometry (ESI-MS). Human serum samples were collected from healthy subjects (n = 10, 20–60 yr) and age-matched ESRD patients (n = 8) before and after HD, purified by affinity chromatography and analyzed by a triple-quadrupole mass spectrometer. The deconvoluted spectra from healthy subjects were all characterized by three peaks attributed to non-glycated mercaptoalbumin (HSA-SH) and to the corresponding adducts with cysteine (HSA-Cys) and glucose (HSA-Glc); relative contents: mercaptoalbumin in both glycated and non-glycated form, HSA-SHt (74 ± 6%), HSA-Cys (26 ± 5%) and HSA-Glc (24 ± 3%). HSA isolated from ESRD patients before HD was characterized by a significant reduction of HSA-SHt (42 ± 7%), and by a concomitant increase of the HSA-Cys adduct (58 ± 7%). Hemodialysis significantly reduced the cysteinylated form (37 ± 7%) and restored HSA-SHt (63 ± 8%) in all the ESRD patients. The mechanism of thiol oxidation and cysteinylation was then studied by mass spectrometry, using LQQCPF as a model peptide and H2O2 as an oxidizing agent.

Flow infusion electrospray ionisation mass spectrometry for high throughput, non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.

Iberian ham typification by direct infusion electrospray and photospray ionization mass spectrometry fingerprinting

Iberian ham is a product of high commercial value whose quality mainly depends on breeding and feeding of pigs in an authorized way. Simple, fast, simple, reliable and high-throughput analytical methods are necessary to assure the quality of ham and for fraud prevention. Tandem mass spectrometry (MS/MS) is proposed as an advantageous alternative over other analytical techniques commonly used in this industry for product authentication. The analytical approach is based on direct infusion electrospray mass spectrometry (ESI(+)-MS) of dichloromethane/methanol (60:40) extracts of ham intramuscular fat. Similarly, atmospheric pressure photoionization ionization mass spectrometry (APPI(+)-MS) was used with a flow injection analysis system for sample introduction with methanol/water (50%) as the mobile phase and toluene as the dopant. All experiments were performed on an API QSTAR® XL Hybrid system using both ESI and APPI sources. The ESI(+)-MS mass spectra present several clusters of peaks attributed to ammonium adducts [M + NH(4) (+) ] of lipid compounds (mono-, di- and triacylglycerols - MGs, DGs, TGs, and free fatty acids - FFAs), that can be identified by MS/MS spectra. On the other hand, the APPI(+)-MS spectra present [M + H(+) ] ions and reflect a higher fragmentation of the sample. Five different types of Iberian ham samples were successfully classified using partial least-squares discriminant analysis (PLS-DA) of data from these samples. The application of direct infusion tandem mass spectrometry to dichloromethane/methanol extracts from intramuscular fat ham allows the simple, fast and reliable fingerprinting typification of different Iberian ham samples from pigs with different diets. With the proposed method, sample handling is minimal and chromatographic separation is not necessary, which represents an evident advantage over other analytical procedures usually used for this purpose.

Diagnosis of lung cancer based on direct-infusion electrospray mass spectrometry of blood plasma metabolites

Lung cancer is one of the most prevalent types of cancer in men and women, and is a leading cause of cancer-related death. Early detection of lung cancer may profoundly reduce cancer death rates. It is therefore extremely important to develop laboratory tests to detect human lung cancer, including at clinically asymptomatic stages. To this end, mass spectrometric metabolite analysis was performed on blood samples collected from patients with lung cancer ( N = 100) and age-matched controls ( N = 100). Proteins were extracted from blood plasma with methanol, and the remaining metabolite fractions were directly analyzed using electrospray ionization (ESI) mass spectrometry. Mass spectra obtained were converted into binary format, aligned, reduced to several variables by principal component analysis (PCA), and finally, classified as cancer cases versus controls by a support vector machine (SVM) algorithm. Repeated random sub-sampling validation revealed an accuracy of classification as high as 93.3% (sensitivity 94.1%, selectivity 92.4%), strongly indicating that direct-infusion ESI mass spectrometry of blood plasma metabolites offers great clinical potential in the diagnosis of early-stage human lung cancer.

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Snake venom peptidomes are valuable sources of pharmacologically active compounds. We analyzed the peptidic fractions (peptides with molecular masses < 10,000 Da) of venoms of Vipera ammodytes meridionalis (Viperinae), the most toxic snake in Europe, and Bothrops jararacussu (Crotalinae), an extremely poisonous snake of South America. Liquid chromatography/mass spectrometry (LC/MS), direct infusion electrospray mass spectrometry (ESI-MS) and matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were applied to characterize the peptides of both snake venoms. 32 bradykinin-potentiating peptides (BPPs) were identified in the Crotalinae venom and their sequences determined. 3 metalloproteinase inhibitors, 10 BPPs and a Kunitz-type inhibitor were observed in the Viperinae venom peptidome. Variability in the C-terminus of homologous BPPs was observed, which can influence the pharmacological effects. The data obtained so far show a subfamily specificity of the venom peptidome in the Viperidae family: BPPs are the major peptide component of the Crotalinae venom peptidome lacking Kunitz-type inhibitors (with one exception) while the Viperinae venom, in addition to BPPs, can contain peptides of the bovine pancreatic trypsin inhibitor family. We found indications for a post-translational phosphorylation of serine residues in Bothrops jararacussu venom BPP (S[combining low line]QGLPPGPPIP), which could be a regulatory mechanism in their interactions with ACE, and might influence the hypotensive effect. Homology between venom BPPs from Viperidae snakes and venom natriuretic peptide precursors from Elapidae snakes suggests a structural similarity between the respective peptides from the peptidomes of both snake families. The results demonstrate that the venoms of both snakes are rich sources of peptides influencing important physiological systems such as blood pressure regulation and hemostasis. The data can be used for pharmacological and medical applications.

Development of a high-performance liquid chromatography-fluorescence detection method for analyzing nonylphenol/dinonylphenol-polyethoxylate-based phosphate esters

A novel reversed phase HPLC method for the simultaneous analysis of surfactants containing nonylphenol/dinonylphenol-polyethoxylates and their o-phosphate esters is reported, in which eluting substances are detected fluorescently. Their chemical structures were elucidated by direct infusion electrospray-mass spectrometry in positive mode. The limits of quantification and range of the method were determined to be 0.1 mg and 0.1–100 mg surfactant L −1, respectively, with a reproducibility (RSD) at a concentration of 38 mg surfactant L −1 of 5.6%. The accuracy was determined by spiking selected process water samples with known amounts of surfactant, and recoveries were typically in the 82–102% range.

Desorption Electrospray Ionization Mass Spectrometry for Trace Analysis of Agrochemicals in Food

Desorption electrospray ionization (DESI) is applied to the rapid, in situ, direct qualitative and quantitative (ultra)trace analysis of agrochemicals in foodstuffs. To evaluate the potential of DESI mass spectrometry (MS) in toxic residue testing in food, 16 representative multiclass agricultural chemicals (pesticides, insecticides, herbicides, and fungicides) were selected (namely, ametryn, amitraz, azoxystrobin, bitertanol, buprofezin, imazalil, imazalil metabolite, isofenphos-methyl, malathion, nitenpyram, prochloraz, spinosad, terbuthylazine, thiabendazole, and thiacloprid). The DESI-MS experiments were performed using 3 microL of solution spotted onto conventional smooth poly(tetrafluoroethylene) (PTFE) surfaces, with examination by MS and tandem mass spectrometry (MS/MS) using an ion trap mass spectrometer. Optimization of the spray solvent led to the use of acetonitrile/water (80:20) (v/v), with 1% formic acid. Most of the compounds tested showed remarkable sensitivity in the positive ion mode, approaching that attainable with conventional direct infusion electrospray mass spectrometry. To evaluate the potential of the proposed approach in real samples, different experiments were performed including the direct DESI-MS/MS analysis of fruit peels and also of fruit/vegetable extracts. The results proved that DESI allows the detection and confirmation of traces of agrochemicals in actual market-purchased samples. In addition, MS/MS confirmation of selected pesticides in spiked vegetable extracts was obtained at absolute levels as low as 1 pg for ametryn. Quantitation of imazalil residues was also undertaken using an isotopically labeled standard. The data obtained were in agreement with those from the liquid chromatography mass spectrometry (LC-MS) reference method, with relative standard deviation (RSD) values consistently below 15%. The results obtained demonstrate the sensitivity of DESI as they meet the stringent European Union pesticide regulation requirements (maximum residue levels) for a large percentage of the studied compounds.

Metabolic profiling by ion mobility mass spectrometry (IMMS)

Ion Mobility Mass Spectrometry (IMMS) was evaluated as an analytical method for metabolic profiling. The specific instrument used in these studies was a direct infusion (DI)-electrospray ionization (ESI)—ambient pressure ion mobility spectrometer (APIMS) coupled to a time-of-flight mass spectrometer (TOFMS). The addition of an ion mobility spectrometer to a mass spectrometer had several advantages over direct infusion electrospray mass spectrometry alone. This tandem instrument (ESI-IMMS) added a rapid separation step with high-resolution prior to mass spectrometric analysis of metabolite mixtures without extending sample preparation time or reducing the high through put potential of direct mass spectrometry. Further, IMMS also reduced the baseline noise common with ESI-MS analyses of complex samples and enabled rapid separation of isobaric metabolites. IMMS was used to analyze the metabolome of Escherichia coli (E. coli), containing a collection of extremely diverse chemical compounds including hydrophobic lipids, inorganic ions, volatile alcohols and ketones, amino and non-amino organic acids, and hydrophilic carbohydrates. IMMS data were collected as two-dimensional spectra showing both mobility and mass of each ion detected. Using direct infusion ESI-IMMS of a non-derivatized methanol extract of an E. coli culture, more than 500 features were detected, of which over 200 intracellular metabolites were tentatively assigned as E. coli metabolites. This analytical method also allowed simultaneous separation of isomeric metabolic features.

Quantitative determination of disaccharide content in digested unfragmented heparin and low molecular weight heparin by direct-infusion electrospray mass spectrometry

Heparins and low molecular weight heparins (LMWHs) are heterogeneous glycosaminoglycans derived from natural sources that are prescribed as anticoagulants. In this work, a direct-infusion electrospray ionization mass spectrometry (ESI-MS) method was applied to the quantitative analysis of known disaccharides in various native heparins and LMWHs after digestion with heparinase enzymes. Disaccharide ΔUA2S → GlcNS6S was found to compose the majority of all samples analyzed (81–88%). The values were significantly higher than those reported by previously published methods. The disaccharide isomer pair ΔUA → GlcNS6S/ΔUA2S → GlcNS was also detected in all samples at lower levels (11–19%). While digestion with heparinases I and II revealed a limited number of disaccharides, the addition of heparinase III to digests led to the detection of disaccharide ΔUA2S → GlcNAc6S in native porcine heparin. This result indicated the importance of utilizing all three heparinases to gain maximum information when analyzing heparin and LMWH digests. This method displayed good between-day (4–6%) and between-digest (1–2%) reproducibility in separate experiments. To determine if the digestion matrix was suppressing the signal of low-abundance disaccharides, several disaccharides were exogenously added at low levels (1–10 pmol/mg) to a quenched digest reaction. Analysis revealed that low level disaccharides were detectable in this matrix above the limits of detection (0.1–0.2 pmol/mg) and quantitation (0.2–0.7 pmol/mg). While this method was unable to distinguish between disaccharide isomers, it utilized simple mass spectrometry instrumentation to provide useful quantitative data for characterizing preparations of native heparin and LMWH, which could be used to compare various marketed preparations of these popular anticoagulants.

Optimizing the connectivity in disulfide-rich peptides: α-conotoxin SII as a case study

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in α-conotoxin SII, of which ∼30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. α-Conotoxin SII was shown to possess 3–8, 2–18, and 4–14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.

Simple Identification of A Cross-Linked Hemoglobin by Tandem Mass Spectrometry in Human Serum

Hemoglobin-based oxygen therapeutics are prepared by reaction of hemoglobin with cross-linking molecules and are utilized as blood substitutes. They can be used as doping agents to increase the oxygen-carrying capacity of hemoglobin. We have compared a glutaraldehyde-polymerized bovine hemoglobin (Oxyglobin, Biopure Corp.) with natural bovine hemoglobin by mass spectrometry in order to detect specific fragment ions of the cross-linked protein for further potential applications in doping control of human blood samples. HCl acid (6 N) hydrolysis was performed in parallel on both proteins. Hydrolysates were then analyzed by direct infusion electrospray mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. Confirmation and precision were obtained by LC-ESIMS(n) experiments performed on an ion trap mass spectrometer. Chromatographic and mass spectrometry data allowed detection of two potential Oxyglobin-specific ions--m/z 299 and 399--that were shown to lose a 159 u neutral fragment under collision-induced dissociation conditions. Thus, monitoring of constant neutral loss of 159 u on acid hydrolysates of human serum samples spiked with different amounts of Oxyglobin has proved to be an efficient screening method to specifically detect and identify Oxyglobin. LC-MS of the spiked serum sample hydrolysates enabled detection of Oxyglobin at a detection limit of 4 g x L(-1).

An optimized protocol for metabolome analysis in yeast using direct infusion electrospray mass spectrometry

A method for the global analysis of yeast intracellular metabolites, based on electrospray mass spectrometry (ES-MS), has been developed. This has involved the optimization of methods for quenching metabolism in Saccharomyces cerevisiae and extracting the metabolites for analysis by positive-ion electrospray mass spectrometry. The influence of cultivation conditions, sampling, quenching and extraction conditions, concentration step, and storage have all been studied and adapted to allow direct infusion of samples into the mass spectrometer and the acquisition of metabolic profiles with simultaneous detection of more than 25 intracellular metabolites. The method, which can be applied to other micro-organisms and biological systems, may be used for comparative analysis and screening of metabolite profiles of yeast strains and mutants under controlled conditions in order to elucidate gene function via metabolomics. Examples of the application of this analytical strategy to specific yeast strains and single-ORF yeast deletion mutants generated through the EUROFAN programme are presented.

Transfer of photosynthate and naturally occurring insecticidal compounds from host plants to the root hemiparasite Santalum acuminatum (Santalaceae)

Plant hemiparasites obtain a wide range of primary compounds from their host plants, including carbon, water and ions. In this paper, we examine the transfer of carbon from the host plant Myoporum parvifolium and the movement of an insecticidal compound from the host Melia azedarach to the root hemiparasite Santalum acuminatum(R.Br) (quandong). By using 14 C we determined that glucose was moving from the M. parvifolium host to the parasite while the carbon fixed by quandongs was found to be mostly in mannitol. Mannitol occurred in fruit, leaf, stem and root tissue and also in xylem sap. We also provide evidence from direct infusion electrospray mass spectrometry (DIEMS) that quandong fruit from trees growing near Melia azedarach (L.) contain an insecticidal compound. This was supported by results from a bioassay in which apple moth (Epiphyas postvittana Wal ker) larvae suffered higher mortality when fed only on quandong fruit that was growing nearM. azedarach than those fed on quandong fruit from trees growing away from M. azedarach.