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Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites

Nature Protocols volume 12, pages 22152231 (2017) | Download Citation

Abstract

Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in 12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.

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Acknowledgements

We thank M. Abu-Remaileh, G. Wyant, N. Kanarek, K. Ottina, and all other members of the D.M.S. lab; we thank E.C. Bayraktar, K. Birsoy, L. Chantranupong, M. Olfky, C. Lewis, B. Chan, T. Kunchok, and H.S. Tsao for their assistance and helpful suggestions. This work was supported by grants from the US National Institutes of Health (R01CA103866, R01CA129105, and R37AI047389) and the Department of Defense (W81XWH-15-1-0230) to D.M.S. W.W.C. was supported by award no. T32GM007753 from the National Institute of General Medical Sciences. The content of this work is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health. D.M.S. is an investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Elizaveta Freinkman

    Present address: Metabolon, Inc., Research Triangle Park, North Carolina, USA.

Affiliations

  1. Department of Biology, Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Walter W Chen
    • , Elizaveta Freinkman
    •  & David M Sabatini
  2. Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Walter W Chen
    •  & David M Sabatini
  3. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.

    • Walter W Chen
    •  & David M Sabatini
  4. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Walter W Chen
    •  & David M Sabatini
  5. Harvard Medical School, Boston, Massachusetts, USA.

    • Walter W Chen

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Contributions

W.W.C. and D.M.S. initiated the project and designed the research. E.F. had an invaluable role in establishing the LC/MS platform and designing the metabolomics methodology. W.W.C. and D.M.S. wrote and edited the manuscript.

Competing interests

W.W.C. is a consultant for VL39, a company developing novel therapeutic modalities for treating mitochondrial pathologies.

Corresponding author

Correspondence to David M Sabatini.

Integrated supplementary information

Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1 and 2.

Excel files

  1. 1.

    Supplementary Data 1

    Comparison of matrix concentrations of mitochondrial metabolites using normal and lengthened isolation times (.xls file).

  2. 2.

    Supplementary Data 2

    List of internal standards used for normalizing various metabolites (.xls file).

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DOI

https://doi.org/10.1038/nprot.2017.104

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