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U13C cell extract of Pichia pastoris – a powerful tool for evaluation of sample preparation in metabolomics

Authors

  • Stefan Neubauer,

    1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
    2. FH Campus Wien, Bioengineering, Vienna, Austria
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  • Christina Haberhauer-Troyer,

    1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Kristaps Klavins,

    1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
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  • Hannes Russmayer,

    1. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    2. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Matthias G. Steiger,

    1. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    2. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Brigitte Gasser,

    1. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    2. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Michael Sauer,

    1. FH Campus Wien, Bioengineering, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    3. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Diethard Mattanovich,

    1. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    2. Department of Biotechnology, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
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  • Stephan Hann,

    1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
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  • Gunda Koellensperger

    Corresponding author
    1. Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB), Vienna, Austria
    • Correspondence: Dr. Gunda Koellensperger, Department of Chemistry, Division of Analytical Chemistry, University of Natural Resources and Life Sciences-BOKU, Muthgasse 18, 1190 Wien, Austria

      E-mail: gunda.koellensperger@boku.ac.at

      Fax: +43147654-6059

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Abstract

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly 13C-labeled (U13C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic 12C and U13C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.

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