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Gunda Koellensperger - One of the best experts on this subject based on the ideXlab platform.
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anion exchange chromatography coupled to high resolution mass spectrometry a powerful tool for merging targeted and non targeted metabolomics
Analytical Chemistry, 2017Co-Authors: Michaela Schwaiger, Evelyn Rampler, Gerrit Hermann, Walter Miklos, Walter Berger, Gunda KoellenspergerAbstract:In this work, simultaneous targeted metabolic profiling by isotope dilution and non-targeted fingerprinting is proposed for cancer cell studies. The novel streamlined metabolomics workflow was established using anion-exchange chromatography (IC) coupled to high-resolution mass spectrometry (MS). The separation time of strong anion-exchange (2 mm column, flow rate 380 μL min–1, injection volume 5 μL) could be decreased to 25 min for a target list comprising organic acids, Sugars, Sugar Phosphates, and nucleotides. Internal standardization by fully 13C labeled Pichia pastoris extracts enabled absolute quantification of the primary metabolites in adherent cancer cell models. Limits of detection (LODs) in the low nanomolar range and excellent intermediate precisions of the isotopologue ratios (on average 0.99) were obtained. Experiments on drug-sensitive versus resi...
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isotopologue analysis of Sugar Phosphates in yeast cell extracts by gas chromatography chemical ionization time of flight mass spectrometry
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Dinh Binh Chu, Gunda Koellensperger, Christina Troyer, Teresa Mairinger, Karin Ortmayr, Stefan Neubauer, Stephan HannAbstract:Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of Sugar Phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of Sugar Phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1–2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6 % and an average bias of 1.4 %.
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speciation analysis of Sugar Phosphates via anion exchange chromatography combined with inductively coupled plasma dynamic reaction cell mass spectrometry optimization for the analysis of yeast cell extracts
Journal of Analytical Atomic Spectrometry, 2014Co-Authors: Dinh Binh Chu, Kristaps Klavins, Gunda Koellensperger, Stephan HannAbstract:Anion exchange chromatography combined with inductively coupled plasma mass spectrometry was introduced and optimized for the separation and accurate quantification of Sugar Phosphates in cell extracts. Sugar Phosphates have been separated on an anion exchange quaternary ammonium functionalized stationary phase (Dionex CarboPAC PA1) via gradient elution with 160 mM Na2CO3 and 50 mM NaOH. After separation, the Sugar Phosphates were on-line transferred to an inductively coupled plasma mass spectrometer after having passed an anion self-regenerating suppressor. Detection was performed via phosphorus as PO+ (m/z 47) was generated by the dynamic reaction cell technique using oxygen as a reaction gas. The ionization suppression process in the inductively coupled plasma caused by the high buffer strength of the chromatographic gradient was assessed by post-column flow injection analysis. It could be shown that the anion self-regenerating suppressor led to a significant reduction of signal suppression. With the new methodology excellent relative standard deviations of retention times of Sugar Phosphates for short- and long-term measurements were achieved. The relative standard deviations of short-term and long-term repeatability of peak areas were below 3.0 and 10.0% respectively. The absolute on-column LODs and LOQs of the optimized method were in the sub-picomole range. The developed method was applied for the purity assessment of commercially available Sugar phosphate standards and evaluated for the quantification of Sugar Phosphates in yeast samples (Pichia pastoris) after extraction with boiling ethanol. Due to the lack of reference materials, the method accuracy was validated by method inter-comparison with an LCxLC-ESI-MS/MS based method, which has been developed in our laboratory.
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fully automated on line two dimensional liquid chromatography in combination with esi ms ms detection for quantification of Sugar Phosphates in yeast cell extracts
Analyst, 2014Co-Authors: Kristaps Klavins, Dinh Binh Chu, Stephan Hann, Gunda KoellenspergerAbstract:A mass spectrometric quantitative assay was developed for the analysis of 10 Sugar Phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten Sugar Phosphates in yeast extracts utilizing internal standardization with a fully labeled 13C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.
Thomas Moritz - One of the best experts on this subject based on the ideXlab platform.
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two step derivatization for determination of Sugar Phosphates in plants by combined reversed phase chromatography tandem mass spectrometry
Plant Methods, 2019Co-Authors: Umut Rende, Totte Niittyla, Thomas MoritzAbstract:Sugar Phosphates are important intermediates of central carbon metabolism in biological systems, with roles in glycolysis, the pentose–phosphate pathway, tricarboxylic acid (TCA) cycle, and many other biosynthesis pathways. Understanding central carbon metabolism requires a simple, robust and comprehensive analytical method. However, Sugar Phosphates are notoriously difficult to analyze by traditional reversed phase liquid chromatography. Here, we show a two-step derivatization of Sugar Phosphates by methoxylamine and propionic acid anhydride after chloroform/methanol (3:7) extraction from Populus leaf and developing wood that improves separation, identification and quantification of Sugar Phosphates by ultra high performance liquid chromatography–electrospray ionization–mass spectrometry (UHPLC–ESI–MS). Standard curves of authentic Sugar Phosphates were generated for concentrations from pg to ng/μl with a correlation coefficient R2 > 0.99. The method showed high sensitivity and repeatability with relative standard deviation (RSD) < 20% based on repeated extraction, derivatization and detection. The analytical accuracy for Populus leaf extracts, determined by a two-level spiking approach of selected metabolites, was 79–107%. The results show the reliability of combined reversed phase liquid chromatography–tandem mass spectrometry for Sugar phosphate analysis and demonstrate the presence of two unknown Sugar Phosphates in Populus extracts.
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MOESM1 of Heterologous phosphoketolase expression redirects flux towards acetate, perturbs Sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
2019Co-Authors: Alexandra Bergman, Thomas Moritz, Jens Nielsen, John Hellgren, Verena Siewers, Yun ChenAbstract:Additional file 1: Table S1. Physiological parameters calculated for control and xfpk(BB) cultivated at pH 4 and 6. Table S2. MRM transitions for LC-QQQ-MS analysis. Table S3. Version number and references for programs used in the NGI-RNAseq pipeline. Figure S1. Quantification of Sugar Phosphates in batch phase. Figure S2. PCA-plot of RNA-sequencing samples. Figure S3. Consensus heat map of gene set analysis
Tetsuro Mimura - One of the best experts on this subject based on the ideXlab platform.
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analysis of Sugar Phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection
Journal of Chromatography A, 2004Co-Authors: Yoko Sekiguchi, Naoto Mitsuhashi, Yoshinori Inoue, Hitoshi Yagisawa, Tetsuro MimuraAbstract:Abstract This paper describes the development of a practical method for the analysis of Sugar Phosphates from the model higher plant Arabidopsis thaliana by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD). The extraction method of Sugar Phosphates from higher plants was first optimized for HPAEC–PAD analysis. In order to improve the resolution in HPAEC–PAD, a column packed with titanium dioxide resin was used. The titanium dioxide column was used as a trap-column for Sugar Phosphates and nucleotides, for the removal of sample matrices. Sample pretreatment was achieved in-line and automatically using a six-port valve placed after the injection valve.
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Sugar Phosphates changes in arabidopsis in response to phosphate nutrition measured by improved ion chromatography with pulsed amperometric detection combined with a titanium dioxide column
Plant Biotechnology, 2004Co-Authors: Yoko Sekiguchi, Naoto Mitsuhashi, Tetsuro MimuraAbstract:We have developed a method for comprehensive analysis of Sugar Phosphates by high performance anion exchange chromatography with pulsed amperometric detection coupled with a titanium dioxide column as a trap-column to remove sample matrices. Levels of Sugar Phosphates and a nucleotide phosphate from Arabidopsis thaliana grown at three different inorganic phosphate (Pi) concentrations in nutrient media or from Pi-related Arabidopsis mutants were investigated. Fructose-6-P, Galactose-1-P, Glucose-1-P, Glucose-6-P and Mannose-6-P apparently increased in proportion to increases in the in vivo level of Pi in wild type plants. In contrast, levels of Sucrose-6-P and UDP-Glucose decreased as the in vivo Pi levels increased. Responses of the former Sugar Phosphates except Mannose-6-P to the in vivo Pi levels in shoots of the mutants were similar in the wild type plants. However, Sucrose-6-P and UDP-Glucose responded differently between the wild type and mutant plants.
Stephan Hann - One of the best experts on this subject based on the ideXlab platform.
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isotopologue analysis of Sugar Phosphates in yeast cell extracts by gas chromatography chemical ionization time of flight mass spectrometry
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Dinh Binh Chu, Gunda Koellensperger, Christina Troyer, Teresa Mairinger, Karin Ortmayr, Stefan Neubauer, Stephan HannAbstract:Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of Sugar Phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of Sugar Phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1–2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6 % and an average bias of 1.4 %.
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speciation analysis of Sugar Phosphates via anion exchange chromatography combined with inductively coupled plasma dynamic reaction cell mass spectrometry optimization for the analysis of yeast cell extracts
Journal of Analytical Atomic Spectrometry, 2014Co-Authors: Dinh Binh Chu, Kristaps Klavins, Gunda Koellensperger, Stephan HannAbstract:Anion exchange chromatography combined with inductively coupled plasma mass spectrometry was introduced and optimized for the separation and accurate quantification of Sugar Phosphates in cell extracts. Sugar Phosphates have been separated on an anion exchange quaternary ammonium functionalized stationary phase (Dionex CarboPAC PA1) via gradient elution with 160 mM Na2CO3 and 50 mM NaOH. After separation, the Sugar Phosphates were on-line transferred to an inductively coupled plasma mass spectrometer after having passed an anion self-regenerating suppressor. Detection was performed via phosphorus as PO+ (m/z 47) was generated by the dynamic reaction cell technique using oxygen as a reaction gas. The ionization suppression process in the inductively coupled plasma caused by the high buffer strength of the chromatographic gradient was assessed by post-column flow injection analysis. It could be shown that the anion self-regenerating suppressor led to a significant reduction of signal suppression. With the new methodology excellent relative standard deviations of retention times of Sugar Phosphates for short- and long-term measurements were achieved. The relative standard deviations of short-term and long-term repeatability of peak areas were below 3.0 and 10.0% respectively. The absolute on-column LODs and LOQs of the optimized method were in the sub-picomole range. The developed method was applied for the purity assessment of commercially available Sugar phosphate standards and evaluated for the quantification of Sugar Phosphates in yeast samples (Pichia pastoris) after extraction with boiling ethanol. Due to the lack of reference materials, the method accuracy was validated by method inter-comparison with an LCxLC-ESI-MS/MS based method, which has been developed in our laboratory.
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fully automated on line two dimensional liquid chromatography in combination with esi ms ms detection for quantification of Sugar Phosphates in yeast cell extracts
Analyst, 2014Co-Authors: Kristaps Klavins, Dinh Binh Chu, Stephan Hann, Gunda KoellenspergerAbstract:A mass spectrometric quantitative assay was developed for the analysis of 10 Sugar Phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten Sugar Phosphates in yeast extracts utilizing internal standardization with a fully labeled 13C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.
Dinh Binh Chu - One of the best experts on this subject based on the ideXlab platform.
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isotopologue analysis of Sugar Phosphates in yeast cell extracts by gas chromatography chemical ionization time of flight mass spectrometry
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Dinh Binh Chu, Gunda Koellensperger, Christina Troyer, Teresa Mairinger, Karin Ortmayr, Stefan Neubauer, Stephan HannAbstract:Metabolic flux analysis is based on the measurement of isotopologue ratios. In this work, a new GC-MS-based method was introduced enabling accurate determination of isotopologue distributions of Sugar Phosphates in cell extracts. A GC-TOFMS procedure was developed involving a two-step online derivatization (ethoximation followed by trimethylsilylation) offering high mass resolution, high mass accuracy and the potential of retrospective data analysis typical for TOFMS. The information loss due to fragmentation intrinsic for isotopologue analysis by electron ionization could be overcome by chemical ionization with methane. A thorough optimization regarding pressure of the reaction gas, emission current, electron energy and temperature of the ion source was carried out. For a substantial panel of Sugar Phosphates both of the glycolysis and the pentose phosphate pathway, sensitive determination of the protonated intact molecular ions together with low abundance fragment ions was successfully achieved. The developed method was evaluated for analysis of Pichia pastoris cell extracts. The measured isotopologue ratios were in the range of 55:1–2:1. The comparison of the experimental isotopologue fractions with the theoretical fractions was excellent, revealing a maximum bias of 4.6 % and an average bias of 1.4 %.
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speciation analysis of Sugar Phosphates via anion exchange chromatography combined with inductively coupled plasma dynamic reaction cell mass spectrometry optimization for the analysis of yeast cell extracts
Journal of Analytical Atomic Spectrometry, 2014Co-Authors: Dinh Binh Chu, Kristaps Klavins, Gunda Koellensperger, Stephan HannAbstract:Anion exchange chromatography combined with inductively coupled plasma mass spectrometry was introduced and optimized for the separation and accurate quantification of Sugar Phosphates in cell extracts. Sugar Phosphates have been separated on an anion exchange quaternary ammonium functionalized stationary phase (Dionex CarboPAC PA1) via gradient elution with 160 mM Na2CO3 and 50 mM NaOH. After separation, the Sugar Phosphates were on-line transferred to an inductively coupled plasma mass spectrometer after having passed an anion self-regenerating suppressor. Detection was performed via phosphorus as PO+ (m/z 47) was generated by the dynamic reaction cell technique using oxygen as a reaction gas. The ionization suppression process in the inductively coupled plasma caused by the high buffer strength of the chromatographic gradient was assessed by post-column flow injection analysis. It could be shown that the anion self-regenerating suppressor led to a significant reduction of signal suppression. With the new methodology excellent relative standard deviations of retention times of Sugar Phosphates for short- and long-term measurements were achieved. The relative standard deviations of short-term and long-term repeatability of peak areas were below 3.0 and 10.0% respectively. The absolute on-column LODs and LOQs of the optimized method were in the sub-picomole range. The developed method was applied for the purity assessment of commercially available Sugar phosphate standards and evaluated for the quantification of Sugar Phosphates in yeast samples (Pichia pastoris) after extraction with boiling ethanol. Due to the lack of reference materials, the method accuracy was validated by method inter-comparison with an LCxLC-ESI-MS/MS based method, which has been developed in our laboratory.
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fully automated on line two dimensional liquid chromatography in combination with esi ms ms detection for quantification of Sugar Phosphates in yeast cell extracts
Analyst, 2014Co-Authors: Kristaps Klavins, Dinh Binh Chu, Stephan Hann, Gunda KoellenspergerAbstract:A mass spectrometric quantitative assay was developed for the analysis of 10 Sugar Phosphates in the yeast Pichia pastoris. As a novelty, two-dimensional chromatography based on a fully automated heart-cutting LC-LC technique was introduced. Using a ten-port valve, ten fractions of the first chromatographic dimension, i.e. anion exchange chromatography (AEC), were transferred and separated by the orthogonal second dimension, i.e. separation on porous graphitized carbon. The chromatographic separation on the second dimension was optimized for each transferred fraction minimizing the separation time and ensuring complete removal of the salt constituents of the AEC eluents. The latter being crucial for electrospray mass spectrometric detection was confirmed by combining the LC-LC separation with on-line ICP-MS detection. These measurements showed that sodium elution was completed after 0.8 min. Consequently, an analysis time of 1 min per transferred peak was established. In this way, the excellent peak capacity given by ion exchange could be conserved in the second dimension at the same time enabling mass spectrometric detection. Sub-μM limits of detection could be obtained by the new LC-LC-MS/MS methods ranging between 0.03 and 0.19 μM for the investigated compounds (only 3GAP showed a LOD of 1 μM). The method was applied to the quantification of ten Sugar Phosphates in yeast extracts utilizing internal standardization with a fully labeled 13C yeast extract. Typically, the standard uncertainties for N = 3 replicates assessed by the LC-LC-MS/MS set-up were <5%.
