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Cell line dependence of metabolite leakage in metabolome analyses of adherent normal and cancer cell lines

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Abstract

Analysis of the metabolome can be sample and cell dependent. In this study, we compared two conventional pre-treatment approaches (trypsinization and cell scraping) in three adherently grown mammalian cell lines (two breast cancer cell lines MDA-MB-436, MCF-7 and an endothelial cell line—HMEC-1). We report experimental evidence, for the first time, demonstrating that metabolite leakage occurs with both treatments, and that the cell lines are differentially influenced. In addition, we examined two recently reported approaches of simultaneous quenching and extraction that showed minimal metabolome leakage. We also investigated the culture of cells on beads for rapid quenching and extraction, as a novel sample handling protocol. For metastatic breast cancer cells MDA-MB-436, the two direct quenching approaches and the bead harvesting approach showed favourable results with respect to metabolome leakage, compared to the conventional approaches. We characterised the recovery of eleven different classes of metabolites identified by gas chromatography–mass spectrometry in the cell extracts and the supernatants following quenching. Analysis of results based on metabolite classes is shown to be a useful approach aiding metabolomic interpretations. We also examined the effect of including a protein precipitation step on the metabolite classes detected. The de-proteinization step did not show significant improvement in overall recoveries. This investigation suggests that it is important to establish the level of metabolome leakage for the specific cell line investigated, irrespective of the methodology employed. Rapid approaches that combine quenching and extraction steps may be more effective in retaining valid metabolome data, with minimal metabolome leakage occurring.

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Abbreviations

GC:

Gas chromatography

MS:

Mass spectrometry

GC–MS:

Gas chromatography–mass spectrometry

LC–MS:

Liquid chromatography–mass spectrometry

NMR:

Nuclear magnetic resonance spectroscopy

TIC:

Total ion current

PCA:

Principal component analysis

AP:

Acetone precipitation

NIST:

National Institute of Standards and Technology

GMD:

Golm metabolome database

AMDIS:

Automated mass spectral deconvolution and identification system

ATCC:

American type culture collection

Da:

Dalton

EDTA:

Ethylene di-amine tetra-acetic acid

MSTFA:

N-Methyl-N-(trimethylsilyl) trifluoroacetamide

LN2:

Liquid nitrogen

AMBIC:

Ammonium bicarbonate

PBS:

Phosphate-buffered saline

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Acknowledgments

Funding support from Chemical Engineering at the Life Science Interface (ChELSI) (EPSRC EP/E036252/1) for part funding of a PhD studentship to RK and the BBSRC DTA (BB/F016840/1) studentship funding to RC are gratefully acknowledged.

Author Contributions

Rahul Vijay Kapoore, Nicola J. Brown and Seetharaman Vaidyanathan conceived and designed the biological study. Rahul Vijay Kapoore and Seetharaman Vaidyanathan designed the metabolomics investigations. Rahul Vijay Kapoore and Rachael Coyle carried out the lab work. Rahul Vijay Kapoore analysed the metabolomics data. Rahul Vijay Kapoore wrote the manuscript with correction and input by all authors. Rachael Coyle contributed to biological experimentation. Seetharaman Vaidyanathan contributed to the statistical assessment of the data. All authors have given approval to the final version of the manuscript.

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Authors and Affiliations

  1. Advanced Biomanufacturing Centre, Department of Chemical and Biological Engineering, ChELSI Institute, The University of Sheffield, Mappin Street, Sheffield, S1 3JD, UK

    Rahul Vijay Kapoore & Seetharaman Vaidyanathan

  2. Microcirculation Research Group, Department of Oncology, Faculty of Medicine, Dentistry and Health, The University of Sheffield, Sheffield, S10 2RX, UK

    Rachael Coyle, Carolyn A. Staton & Nicola J. Brown

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  1. Rahul Vijay Kapoore
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  2. Rachael Coyle
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  3. Carolyn A. Staton
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  5. Seetharaman Vaidyanathan
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Correspondence to Seetharaman Vaidyanathan.

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Kapoore, R.V., Coyle, R., Staton, C.A. et al. Cell line dependence of metabolite leakage in metabolome analyses of adherent normal and cancer cell lines. Metabolomics 11, 1743–1755 (2015). https://doi.org/10.1007/s11306-015-0833-4

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  • DOI: https://doi.org/10.1007/s11306-015-0833-4

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