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A positive/negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue

Nature Protocols volume 7, pages 872–881 (2012)Cite this article

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Abstract

The revival of interest in cancer cell metabolism in recent years has prompted the need for quantitative analytical platforms for studying metabolites from in vivo sources. We implemented a quantitative polar metabolomics profiling platform using selected reaction monitoring with a 5500 QTRAP hybrid triple quadrupole mass spectrometer that covers all major metabolic pathways. The platform uses hydrophilic interaction liquid chromatography with positive/negative ion switching to analyze 258 metabolites (289 Q1/Q3 transitions) from a single 15-min liquid chromatography–mass spectrometry acquisition with a 3-ms dwell time and a 1.55-s duty cycle time. Previous platforms use more than one experiment to profile this number of metabolites from different ionization modes. The platform is compatible with polar metabolites from any biological source, including fresh tissues, cancer cells, bodily fluids and formalin-fixed paraffin-embedded tumor tissue. Relative quantification can be achieved without using internal standards, and integrated peak areas based on total ion current can be used for statistical analyses and pathway analyses across biological sample conditions. The procedure takes ∼12 h from metabolite extraction to peak integration for a data set containing 15 total samples (∼6 h for a single sample).

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Figure 1: Metabolomics platform schematic.
Figure 2: Reproducibility and heat maps from cancer cell metabolomics.
Figure 3: In vivo metabolomics profiling.

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Acknowledgements

We thank M. Vander Heiden, L. Cantley, J. Rabinowitz, J. Locasale, C. Lyssiotis, E. Driggers, L. Peshkin, R. Rahal and A. Sasaki for helpful and informative philosophical and technical discussions. We also thank C.-L. Wu and E. Wong for providing FFPE and CSF samples, respectively. This research was supported by grants from the US National Institutes of Health 5P01CA120964 and 5P30CA006516 (J.M.A.) and the Beth Israel Deaconess Medical Center Research Capital Fund for funding the mass spectrometry instrumentation (J.M.A.).

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Author notes

  1. Min Yuan and Susanne B Breitkopf: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Signal Transduction/Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

    Min Yuan, Susanne B Breitkopf, Xuemei Yang & John M Asara

  2. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    John M Asara

Authors
  1. Min Yuan
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Contributions

J.M.A. developed the platform and wrote the protocol. M.Y. and S.B.B. optimized and edited the protocol. X.Y., S.B.B. and M.Y. prepared biological samples for testing the protocol. J.M.A., M.Y. and S.B.B. performed data analysis.

Corresponding author

Correspondence to John M Asara.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

A list of 258 unique polar metabolites and 289 Q1/Q3 SRM transitions for positive/negative ion switching along with collision energies (CE) and dwell time on the 5500 QTRAP for the targeted metabolites. Chemical formulas and database identifiers (KEGG, HMDB, or PubChem) for pathway analysis are also included in the table. (XLS 62 kb)

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Yuan, M., Breitkopf, S., Yang, X. et al. A positive/negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue. Nat Protoc 7, 872–881 (2012). https://doi.org/10.1038/nprot.2012.024

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