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Fast sampling method for mammalian cell metabolic analyses using liquid chromatography–mass spectrometry

Nature Protocols volume 10, pages 1–11 (2015)Cite this article

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Abstract

Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a −20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

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Figure 1: Flowchart of different protocol steps.
Figure 2: Washing of cells cultivated on a cover glass.
Figure 3: Optical inspection of HeLa cells.
Figure 4: Evaluation of ion chromatograms upon LC-MS.
Figure 5: Extracted ion chromatograms of selected metabolites from washed HeLa sample.

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Acknowledgements

Funding was provided by SystemsX, The Swiss Initiative in Systems Biology (Research, Technology and Development (RTD) project 'BattleX').

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Author notes

  1. Giuseppe Martano and Nathanaël Delmotte: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Institute of Microbiology, ETH Zurich, Zurich, Switzerland

    Giuseppe Martano, Nathanaël Delmotte, Patrick Kiefer, Philipp Christen & Julia A Vorholt

  2. Biozentrum, University of Basel, Basel, Switzerland

    David Kentner & Dirk Bumann

Authors
  1. Giuseppe Martano
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  2. Nathanaël Delmotte
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  3. Patrick Kiefer
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  7. Julia A Vorholt
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Contributions

All authors developed the protocol and discussed its applications. G.M., N.D., P.C. and D.K. acquired the data for setup experiments. G.M. and N.D. analyzed the data. P.K. wrote the manuscript with contributions from G.M., P.C. and D.K. All authors commented on the manuscript and approved it.

Corresponding author

Correspondence to Julia A Vorholt.

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The authors declare no competing financial interests.

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Martano, G., Delmotte, N., Kiefer, P. et al. Fast sampling method for mammalian cell metabolic analyses using liquid chromatography–mass spectrometry. Nat Protoc 10, 1–11 (2015). https://doi.org/10.1038/nprot.2014.198

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