High-performance metabolic profiling with dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) for study of the exposome

Studies of gene–environment (G × E) interactions require effective characterization of all environmental exposures from conception to death, termed the exposome. The exposome includes environmental exposures that impact health. Improved metabolic profiling methods are needed to characterize these exposures for use in personalized medicine. In the present study, we compared the analytic capability of dual chromatography-Fourier-transform mass spectrometry (DC-FTMS) to previously used liquid chromatography-FTMS (LC-FTMS) analysis for high-throughput, top-down metabolic profiling. For DC-FTMS, we combined data from sequential LC-FTMS analyses using reverse phase (C18) chromatography and anion exchange (AE) chromatography. Each analysis was performed with electrospray ionization in the positive ion mode and detection from m/z 85 to 850. Run time for each column was 10 min with gradient elution; 10 μl extracts of plasma from humans and common marmosets were used for analysis. In comparison to analysis with the AE column alone, addition of the second LC-FTMS analysis with the C18 column increased m/z feature detection by 23–36%, yielding a total number of features up to 7,000 for individual samples. Approximately 50% of the m/z matched to known chemicals in metabolomic databases, and 23% of the m/z were common to analyses on both columns. Database matches included insecticides, herbicides, flame retardants, and plasticizers. Modularity clustering algorithms applied to MS-data showed the ability to detection clusters and ion interactions. DC-FTMS thus provides improved capability for high-performance metabolic profiling of the exposome and development of personalized medicine.     

Metabolome-Wide Association Study of Neovascular Age-Related Macular Degeneration

Purpose

To determine if plasma metabolic profiles can detect differences between patients with neovascular age-related macular degeneration (NVAMD) and similarly-aged controls.

Methods

Metabolomic analysis using liquid chromatography with Fourier-transform mass spectrometry (LC-FTMS) was performed on plasma samples from 26 NVAMD patients and 19 controls. Data were collected from mass/charge ratio (m/z) 85 to 850 on a Thermo LTQ-FT mass spectrometer, and metabolic features were extracted using an adaptive processing software package. Both non-transformed and log2 transformed data were corrected using Benjamini and Hochberg False Discovery Rate (FDR) to account for multiple testing. Orthogonal Partial Least Squares-Discriminant Analysis was performed to determine metabolic features that distinguished NVAMD patients from controls. Individual m/z features were matched to the Kyoto Encyclopedia of Genes and Genomes database and the Metlin metabolomics database, and metabolic pathways associated with NVAMD were identified using MetScape.

Results

Of the 1680 total m/z features detected by LC-FTMS, 94 unique m/z features were significantly different between NVAMD patients and controls using FDR (q = 0.05). A comparison of these features to those found with log2 transformed data (n = 132, q = 0.2) revealed 40 features in common, reaffirming the involvement of certain metabolites. Such metabolites included di- and tripeptides, covalently modified amino acids, bile acids, and vitamin D-related metabolites. Correlation analysis revealed associations among certain significant features, and pathway analysis demonstrated broader changes in tyrosine metabolism, sulfur amino acid metabolism, and amino acids related to urea metabolism.

Conclusions

These data suggest that metabolomic analysis can identify a panel of individual metabolites that differ between NVAMD cases and controls. Pathway analysis can assess the involvement of certain metabolic pathways, such as tyrosine and urea metabolism, and can provide further insight into the pathophysiology of AMD.     

Enhancement of ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by combining resin HP20 addition and metabolic profiling analysis

Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.     

A novel approach combining the Calgary Biofilm Device and Phenotype MicroArray for the characterization of the chemical sensitivity of bacterial biofilms

A rapid method for screening the metabolic susceptibility of biofilms to toxic compounds was developed by combining the Calgary Biofilm Device (MBEC device) and Phenotype MicroArray (PM) technology. The method was developed using Pseudomonas alcaliphila 34, a Cr(VI)-hyper-resistant bacterium, as the test organism. P. alcaliphila produced a robust biofilm after incubation for 16 h, reaching the maximum value after incubation for 24 h (9.4 × 10⁶ ± 3.3 × 10⁶ CFU peg^?1). In order to detect the metabolic activity of cells in the biofilm, dye E (5×) and menadione sodium bisulphate (100 μM) were selected for redox detection chemistry, because they produced a high colorimetric yield in response to bacterial metabolism (340.4 ± 6.9 Omnilog Arbitrary Units). This combined approach, which avoids the limitations of traditional plate counts, was validated by testing the susceptibility of P. alcaliphila biofilm to 22 toxic compounds. For each compound the concentration level that significantly lowered the metabolic activity of the biofilm was identified. Chemical sensitivity analysis of the planktonic culture was also performed, allowing comparison of the metabolic susceptibility patterns of biofilm and planktonic cultures.     

Bioavailability and metabolism of fucoxanthin in rats: structural characterization of metabolites by LC-MS (APCI)

This study reports bioavailability and metabolism of fucoxanthin (FUCO) from brown algae Padina tetrastromatica in rats. Rats were divided into two groups (n = 25/group). Group one was fed basal diet (control) while the group two received retinol deficient diet (RD group) for 8 weeks. After confirmed RD in blood (0.53 μmol/l), rats were further sub-grouped (n = 5/sub group), intubated a dose of FUCO (0.83 μmol) and killed after 0, 2, 4, 6 and 8 h. The plasma levels (area under curve/8 h) of FUCO (fucoxanthinol (FUOH) + amarouciaxanthin (AAx)) was 2.93 (RD group) and 2.74 pmol/dl (control), respectively. No newly formed retinol was detected in RD rats intubated with FUCO. Besides FUOH (m/z 617 (M+H)⁺) and AAx (m/z 617 (M+H^?)⁺), other deacetylated, hydrolyzed and demethylated metabolites of bearing molecular mass at m/z 600.6 (FUOH–H₂O), m/z 597 (AAx–H₂O), m/z 579 (AAx–2H₂O+1), m/z 551 (AAx–2H₂O–2CH₃+2) and m/z 523 (AAx–2H₂O–4CH₃+4) were also detected in plasma and liver by LC-MS (APCI). Although biological functions of FUCO metabolites need thorough investigation, this is the first detailed report on FUCO metabolites in rats.     

Determination of Concentration of Methotrexate Enantiomers in Intracellular and Extracellular Fluids of HepG2 Cells by Liquid Chromatography–Tandem Mass Spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry assay (LC–MS/MS) with electrospray ionization was developed and validated for the quantitative determination of the concentration of methotrexate (MTX) enantiomers in intracellular and extracellular fluids of HepG₂ cells. The analytes were extracted from homogenates using organic solvent to precipitate proteins. The extracted samples were analyzed by LC–MS/MS, operating in multiple reactions monitoring (MRM) mode. The condition of HPLC included the following: Gemini column (3 μm, 3.0 × 75 mm) with chromatographic column was used, and the mobile phase consisting of gradient elution utilized 0.1 % formic acid as solvent A and acetonitrile as solvent B at a flow rate of 0.4 mL min^?1. The gradient was as follows: 0–7.0 min 10–90 % B, 7.0–10 min 90 % B followed by 3 min. The column temperature was maintained at 40 °C. The condition of MS included using electrospray ionization source; MRM mode with the transitions of m/z 455.2 → m/z 308.1 was used to quantify MTX enantiomers. The linear calibration curve was obtained in the concentration range of 10.0 to 10,000 ng mL^?1 for MTX enantiomers in intracellular and extracellular fluids. The inter- and intraday precision was less than 15 %. The mean recovery of (+)-MTX and (?)-MTX in the extracellular fluid of HepG₂ cells were 95.30 and 96.53 %, respectively, and the mean recovery of (+)-MTX and (?)-MTX in the intracellular fluid of HepG2 cells were 93.53 and 94.12 %, respectively. This method was successfully used to detect the concentration of MTX enantiomers in the intracellular and extracellular fluids of HepG₂ cells and that the concentration of (+)-MTX in intracellular fluid was twice higher than the concentration of (?)-MTX in intracellular fluid. The inhibitory effect of (+)-MTX and (?)-MTX was (+)-MTX > (?)-MTX. It is a simple, precise method that can effectively explain the difference in pharamocological effect of MTX enantiomers in vitro.     

Effect of a combination of inulin and polyphenol-containing adzuki bean extract on intestinal fermentation in vitro and in vivo

The effect of a combination of inulin (INU) and polyphenol-containing adzuki bean extract (AE) on intestinal fermentation was examined in vitro using fermenters for 48 h and in vivo using rats for 28 d. The total short-chain fatty acid concentrations in the fermenters were decreased by a combination of INU and AE, but the concentration in the INU + AE group was higher than the cellulose (CEL) and CEL + AE groups. The cecal propionate concentration was increased by a combination of INU and AE compared with their single supplement. The ammonia-nitrogen concentration in the fermenters and rat cecum was decreased by INU and AE. Cecal mucin levels were increased by INU and AE respectively. Therefore, our observations suggested that the combination of INU and AE might be a material of functional food that includes several healthy effects through intestinal fermentation.     

A combination of ultrasonic-assisted extraction with RRLC-QQQ method for the determination of artemisinin in the Chinese herb Artemisia annua L

Introduction – Artemisinin, the primary active ingredient of the Chinese herb Artemisia annua L., is known to have considerable anti‐malaria properties. However, rapid, sensitive and selective method for the determination of artemisinin in it is not currently available. Objective – To develop and validate an efficient method for extraction and analysis of artemisinin from the plant samples of Artemisia annua L. by rapid resolution liquid chromatography triple quadrupole mass spectrometry (RRLC‐QQQ). Methodology – Following ultrasound‐assisted extraction (USE), RRLC‐QQQ was utilised to separate and determine artemisinin from the plant sample of Artemisia annua L. The LC separation, QQQ‐MS detection and multiple reaction monitoring (MRM) mode were optimised, and the method validation concluding selectivity, calibration, accuracy and precision, and recovery were also evaluated. Results – LC separation was performed with an isocratic elution of 20% of methanol–water (10 mmol/L ammonium acetate, pH 4.0) on a C₁₈ column. The triple quadrupole MS detection was carried out under MRM mode of precursor ion [M + H]⁺ → fragment ions m/z 265.1 and m/z 247.2. The limits of detection and quantitation of artemisinin were 0.20 and 0.75 ng/mL, respectively. The intra‐ and inter‐day precisions did not exceed 3.71%, and the deviation of the intra‐ and inter‐day mean values did not exceed ±7.50. The average recoveries for artemisinin ranged from 92.45 to 103.8% with an RSD from 2.47 to 2.79%. Conclusion – The developed RRLC‐QQQ assay is an efficient method for separation and determination of artemisinin from the plant samples of Artemisia annua L. Copyright © 2011 John Wiley & Sons, Ltd.     

Population-level study links short-wavelength nighttime illumination with breast cancer incidence in a major metropolitan area

ABSTRACT

Several population-level studies revealed a positive association between breast cancer (BC) incidence and artificial light at night (ALAN) exposure. However, the effect of short-wavelength illumination, implicated by laboratory research and small-scale cohort studies as the main driving force behind BC–ALAN association, has not been supported by any population-level study carried out to date. We investigated a possible link between BC and ALAN of different subspectra using a multi-spectral year-2011 satellite image, taken from the International Space Station, and superimposing it with year-2013 BC incidence data available for the Great Haifa Metropolitan Area in Israel. The analysis was performed using both ordinary least square (OLS) and spatial dependency models, controlling for socioeconomic and locational attributes of the study area. The study revealed strong associations between BC and blue and green light subspectra (B = 0.336 ± 0.001 and B = 0.335 ± 0.002, respectively; p < 0.01), compared to a somewhat weaker effect for the red subspectrum (B = 0.056 ± 0.001; p < 0.01). However, spatial dependency models, controlling for spatial autocorrelation of regression residuals, confirmed only a positive association between BC incidence and short-wavelength (blue) ALAN subspectrum (z = 2.462, p < 0.05) while reporting insignificant associations between BC and either green (z = 1.425, p > 0.1) or red (z = ?0.604, p > 0.1) subspectra. The obtained result is in line with the results of laboratory- and small-scale cohort studies linking short-wavelength nighttime illumination with circadian disruption and melatonin suppression. The detected effect of blue lights on BC incidence may help to develop informed illumination policies aimed at minimizing the adverse health effects of ALAN exposure on human health.     

Metabolic profiling of cervical tubercular lymphadenitis tissues by proton HR-MAS NMR spectroscopy

Proton metabolic profiling of incisional biopsied cervical lymph node tissue specimens of 109 patients suffering from tubercular (CTBL) and non-specific (NSCLA) lymphadenitis were analyzed by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. In the present study, 40 endogenous metabolites namely, myo-inositol (m-Ins), branched chain amino acids (BCAA), glutamate, serine, taurine (Tau) aromatic amino acids, choline (Cho) containing compounds and glucose were characterized. To the best of our knowledge, this is the first report on metabolic profiling of cervical tubercular lymph node tissues using HR-MAS NMR spectroscopy. The principal component analysis revealed a clear discrimination between CTBL and NSCLA tissues. Increase in the concentration of mobile poly unsaturated fatty acids, BCAA, Cho, Tau, glycine and a decrease in the concentration of lactate, phosphocholine and m-Ins was observed in CTBL cases. The partial least square discriminant analysis (PLS-DA) with R ² = 0.95 and Q ² = 0.92 provided >98 % of correct classification between the two groups. A PLS-DA training set model of 75 % (CTBL = 54, NSCLA = 27) of the subjects when subjected for prediction of 25 % cases (CTBL = 18, NSCLA = 10) as an unknown dataset provided more than 98 % of diagnostic accuracy in their respective histological categories. The receiver operator characteristic curve was generated from PLS-DA factor-1 projected an area under the curve of 0.962. The metabolic profile obtained from HR-MAS NMR spectroscopy may be used as surrogate markers in vivo MRS for differentiating between CTBL and NSCLA cases non-invasively.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

Comparative metabolic profiling-based improvement of rapamycin production by Streptomyces hygroscopicus

Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds.     

Simultaneous determination of norethisterone and six metabolites in human plasma by capillary gas chromatography with mass-selective detection

A method for the simultaneous determination of norethisterone (NET) and six metabolites in human plasma by capillary gas chromatography-mass-selective detection (GC-MS) is described. The compounds are determined in plasma after enzymatic hydrolysis. After addition of norgestrel as the internal standard, the compounds are extracted from plasma at pH 5 using an Extrelut column and elution with dichloromethane. After evaporation, the compounds are converted into bistrimethylsilyl derivatives which are determined by gas chromatography using a mass-selective detector at m/z 429 for the two dihydro-NET (5β-NET and 5α-NET), m/z 431 for the four tetrahydro-NET (3α,5α-NET, 3α,5β-NET, 3β,5β-NET and 3β,5α-NET), m/z 442 for NET and m/z 456 for the internal standard. The reproducibility and accuracy of the method were found suitable over the range of concentrations between 0.50 and 8 ng/ml for NET, and metabolites except for 5α-dihydro-NET (between 1 and 8 ng/ml). The method was applied to clinical samples.     

Identification of novel serum peptides biomarkers for female breast cancer patients in Western China

This study aimed to identify novel serum peptides biomarkers for female breast cancer (BC) patients. We analyzed the serum proteomic profiling of 247 serum samples from 96 BC patients, 48 additional paired pre‐ and postoperative BC patients, 39 fibroadenoma patients as benign disease controls, and 64 healthy controls, using magnetic‐bead‐based separation followed by MALDI‐TOF MS. ClinProTools software identified 78 m/z peaks that differed among all analyzed groups, ten peaks were significantly different (P < 0.0001), with Peaks 1–6 upregulated and Peaks 7–10 downregulated in BC. Moreover, three peaks of ten (Peak 1, m/z: 2660.11; Peak 2, m/z: 1061.09; Peak 10, m/z: 1041.25) showed a tendency to return to healthy control values after surgery. And these three peptide biomarkers were identified as FGA605‐629, ITIH4 347–356, and APOA2 43–52. Methods used in this study could generate serum peptidome profiles of BC, and provide a new approach to identify potential biomarkers for diagnosis as well as prognosis of this malignancy.     

Plant lipidomics based on hydrophilic interaction chromatography coupled to ion trap time-of-flight mass spectrometry

Plants synthesize a wide range of hydrophobic compounds, generally known as lipids. Here, we report an application of liquid chromatography ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS) for plant lipidomics. Using hydrophilic interaction chromatography (HILIC) for class separation, typical membrane lipids including glycerolipids, steryl glucosides and glucosylceramides, and hydrophobic plant secondary metabolites such as saponins were analyzed simultaneously. By this method, we annotated approximately 100 molecules from Arabidopsis thaliana. To demonstrate the application of this method to biological study, we analyzed Arabidopsis mutant trigalactosyldiacylglycerol3 (tgd3), which has a complex metabolic phenotype including the accumulation of unusual forms of galactolipids. Lipid profiling by LC-MS revealed that tgd3 accumulated an unusual form of digalactosyldiacylglycerol, annotated as Gal(β1 → 6)βGalDG. The compositional difference between normal and unusual forms of digalactosyldiacylglycerol was detected by this method. In addition, we analyzed well-known Arabidopsis mutants ats1-1, fad6-1, and fad7-2, which are also disrupted in lipid metabolic genes. Untargeted lipidome analysis coupled with multivariate analysis clearly discriminated the mutants and their distinctive metabolites. These results indicated that HILIC-MS is an efficient method for plant lipidomics.

     

Determination of acrolein in human urine by headspace gas chromatography and mass spectrometry

A rapid and sensitive headspace gas chromatographic and mass spectrometric (GC–MS) method was developed for the determination of acrolein in human urine. A 0.5-ml urine sample in a glass vial containing propionaldehyde as an internal standard was heated at 80°C for 5 min. A 0.1-ml volume of headspace vapor was injected into a GC–MS instrument. Acrolein and propionaldehyde were coeluted at 3.1 min using a DB-1 capillary column, and well separated by selective ion monitoring (SIM) mode using ions m/z 56.05 and m/z 58.05. The interassay and intraassay coefficient of variation were 0.99% and 3.3%. The calibration curve demonstrated a good linearity throughout concentrations ranging from 1 to 1000 nM. However, due to a wide variation of acrolein evaporation rates from human urine, a calibration curve must be established for each urine specimen using a standard addition method and detection limit varied from 1 to 5 nM. The total analysis time for two samples from one urine specimen required about 15 min. Therefore, this method is convenient for the urgent monitoring of urinary acrolein in patients to whom alkylating agents are administered.     

Lipopeptides Produced by a Soil <Emphasis Type="Italic">Bacillus Megaterium</Emphasis> Strain

A soil microorganism identified as Bacillum megaterium was found to produce several antibiotics substances after growth for 20 h at 37°C in a mineral culture medium. Analysis both by electron spray ionization (ESI) and matrix-assisted laser desorption ionization—time of flight (MALDI-TOF) mass spectrometry (MS) identified these substances as lipopeptides. Predominant peaks at m/z 1,041 and m/z 1,065 revealed ions which are compatible with surfactins and lichenysins, respectively. Two other ions m/z 1,057 and m/z 1,464 were further studied by collision-induced dissociation (CID) unveiling an iturin A at the first and fengycins A and B at the second m/z peaks. The CID spectrum of the m/z 1,464 ion also suggests the existence of fengycins A and B variants in which Ile was changed to Val in the position 10 of the peptide moiety. Raw mixtures of all these compounds were also assayed for antibiotic features. The data enlighten the unusual diversity of the lipopeptide mixture produced by a sole Bacillus species.     

A search for glomuferrin: a potential siderophore of arbuscular mycorrhizal fungi of the genus <Emphasis Type="Italic">Glomus</Emphasis>

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2–3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H]⁺ with a relative mass error of ? = 0.47 ppm an elemental composition of C₁₆H₂₁N₂O₁₀ [M+H]⁺ was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C₁₆H₁₉N₂O₁₀Fe (? = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).     

Balancing robust quantification and identification for iTRAQ: Application of UHR‐ToF MS

iTRAQ reagents allow the simultaneous multiplex identification and quantification of a large number of proteins. Success depends on effective peptide fragmentation in order to generate both peptide sequence ions (higher mass region, 150–2200 m/z) and reporter ions (low mass region, 113–121 m/z) for protein identification and relative quantification, respectively. After collision‐induced dissociation, the key requirements to achieve a good balance between the high and low m/z ions are effective ion transmission and detection across the MS/MS mass range, since the ion transmission of the higher m/z range competes with that of the low m/z range. This study describes an analytical strategy for the implementation of iTRAQ on maXis UHR‐Qq‐ToF instruments, and discusses the impact of adjusting the MS/MS ion transmission parameters on the quality of the overall data sets. A technical discussion highlights a number of maXis‐specific parameters, their impact of quantification and identification, and their cross‐interactions.     

Karyotype analysis and DNA content in some species of Chrysolaena (Vernonieae,Asteraceae)

Cytogenetic characterization by karyotyping and determination of DNA content by flow cytometry of five species of Chrysolaena (Vernonieae, Asteraceae) was performed. This is the first study of nuclear DNA content realized in the genus. The 2C-values were compared with the ploidy level and the total karyotype length (TKL) of each species. Mitotic analysis revealed a base chromosome number x = 10 for all entities and different ploidy levels, from diploid (2n = 2x = 20) to octoploid (2n = 8x = 80). All species showed bimodal karyotypes composed of metacentric and submetacentric chromosomes. The average chromosome size (ML) varied from 1.86 μm to 2.70 μm, while the TKL ranged from 18.65 μm to 80.55 μm. The intrachromosomal asymmetry index (A₁) varied from 0.27 to 0.38, while the interchromosomal asymmetry index (A₂) ranged from 0.19 to 0.25. A new cytotype is reported for the first time for C. propinqua. Accessory chromosomes found in C. verbascifolia, C. cognata, C. flexuosa, and C. propinqua are also reported as new.