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Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells

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Abstract

In this study, a liquid chromatography mass spectrometry (LC/MS)-based metabolomics protocol was optimized for quenching, harvesting, and extraction of metabolites from the human pancreatic cancer cell line Panc-1. Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in water were compared for sample harvesting. Four different extraction methods were compared to investigate the efficiency of intracellular metabolite extraction, including pure acetonitrile, methanol, methanol/chloroform/H2O, and methanol/chloroform/acetonitrile. The separation efficiencies of hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) with UPLC-QTOF-MS were also evaluated. Global metabolomics profiles were compared; the number of total detected features and the recovery and relative extraction efficiencies of target metabolites were assessed. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Direct scraping after flash quenching with liquid nitrogen was chosen to harvest Panc-1 cells which allowed for samples to be stored before extraction. Methanol/chloroform/H2O was chosen as the optimal extraction solvent to recover the highest number of intracellular features with the best reproducibility. HILIC had better resolution for intracellular metabolites of Panc-1 cells. This optimized method therefore provides high sensitivity and reproducibility for a variety of cellular metabolites and can be applicable to further LC/MS-based global metabolomics study on Panc-1 cell lines and possibly other cancer cell lines with similar chemical and physical properties.

Optimized harvesting, extraction and analytical protocols for cell metabolomics analysis.

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Acknowledgments

This study was supported by the National Cancer Institute Intramural Research Program and National Institutes of Health grant ES022186. We thank the Natural Science Foundation of China for collaborative research for a grant to Dr. H.C.Bi. (grant No. 81001685).

Conflict of interest

The authors have no conflict of interest.

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Authors and Affiliations

  1. School of Pharmaceutical Sciences, Sun Yat-sen University, 132# Waihuandong Road, Guangzhou University City, Guangzhou, 510006, China

    Huichang Bi

  2. Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD, 20892, USA

    Huichang Bi, Kristopher W. Krausz, Soumen K. Manna, Fei Li, Caroline H. Johnson & Frank J. Gonzalez

  3. Center for Metabolomics and Mass Spectrometry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA

    Caroline H. Johnson

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  1. Huichang Bi
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  2. Kristopher W. Krausz
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  3. Soumen K. Manna
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  4. Fei Li
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Correspondence to Huichang Bi.

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Bi, H., Krausz, K.W., Manna, S.K. et al. Optimization of harvesting, extraction, and analytical protocols for UPLC-ESI-MS-based metabolomic analysis of adherent mammalian cancer cells. Anal Bioanal Chem 405, 5279–5289 (2013). https://doi.org/10.1007/s00216-013-6927-9

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  • DOI: https://doi.org/10.1007/s00216-013-6927-9

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