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Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols

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Abstract

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid–base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid–base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.

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Acknowledgement

This study was funded in part by BayGene and the intramural ReForM C program of the Regensburg School of Medicine. The help of Dr. Y. Reinders, Regina Lindner, and Ruth Spaeth is gratefully acknowledged.

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Authors and Affiliations

  1. Institute of Functional Genomics, University of Regensburg, Josef-Engert-Str. 9, 93053 Regensburg, Germany

    Katja Dettmer, Nadine Nürnberger, Hannelore Kaspar, Martin F. Almstetter & Peter J. Oefner

  2. Department of Anesthesiology, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany

    Michael A. Gruber

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  1. Katja Dettmer
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  2. Nadine Nürnberger
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  3. Hannelore Kaspar
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  4. Michael A. Gruber
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  5. Martin F. Almstetter
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  6. Peter J. Oefner
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Correspondence to Katja Dettmer.

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Dettmer, K., Nürnberger, N., Kaspar, H. et al. Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols. Anal Bioanal Chem 399, 1127–1139 (2011). https://doi.org/10.1007/s00216-010-4425-x

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  • DOI: https://doi.org/10.1007/s00216-010-4425-x

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