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Isotopologue analysis of sugar phosphates in yeast cell extracts by gas chromatography chemical ionization time-of-flight mass spectrometry

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Abstract

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 %.

Isotopologue distribution of glucose-6-phosphate measured by GC-QTOFMS after derivatization

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Acknowledgments

Dinh Binh Chu gratefully acknowledges Erasmus Mundus Action 2 (www.eurasia2.cz) for financial support. EQ BOKU Vienna is acknowledged for providing mass spectrometric instrumentation. This work has been supported by the Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT through the Austrian FFG-COMET-Funding Program.

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Authors and Affiliations

  1. Division of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences, BOKU Vienna, Muthgasse 18, 1190, Vienna, Austria

    Dinh Binh Chu, Teresa Mairinger, Karin Ortmayr, Stefan Neubauer & Stephan Hann

  2. Austrian Center of Industrial Biotechnology, 1190, Vienna, Austria

    Christina Troyer, Teresa Mairinger, Stefan Neubauer, Gunda Koellensperger & Stephan Hann

  3. Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Waehringer 42, 1090, Vienna, Austria

    Gunda Koellensperger

  4. Department of Analytical Chemistry, School of Chemical Engineering, Hanoi University of Science and Technology, No 1 Dai Co Viet Road, Hanoi, Vietnam

    Dinh Binh Chu

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  1. Dinh Binh Chu
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Correspondence to Stephan Hann.

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Chu, D.B., Troyer, C., Mairinger, T. et al. Isotopologue analysis of sugar phosphates in yeast cell extracts by gas chromatography chemical ionization time-of-flight mass spectrometry. Anal Bioanal Chem 407, 2865–2875 (2015). https://doi.org/10.1007/s00216-015-8521-9

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  • DOI: https://doi.org/10.1007/s00216-015-8521-9

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