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

Nature Protocols volume 12, pages 2215–2231 (2017)Cite this article

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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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Figure 1: Workflow for quantifying matrix concentrations of mitochondrial metabolites.
Figure 2: The degree of HA-MITO construct expression affects the purity of isolated mitochondria.
Figure 3: Quantitative benchmarks for assessing reliability of data.

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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.

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Author notes

  1. Elizaveta Freinkman

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

Authors and Affiliations

  1. Department of Biology,

    Walter W Chen, Elizaveta Freinkman & David M Sabatini

  2. Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Walter W Chen, Elizaveta Freinkman & David M Sabatini

  3. Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Walter W Chen & David M Sabatini

  4. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA

    Walter W Chen & David M Sabatini

  5. Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Walter W Chen & David M Sabatini

  6. 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.

Corresponding author

Correspondence to David M Sabatini.

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Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Gating strategy for flow cytometric analyses.

Wildtype HeLa cells (i.e., no viral transduction with Control-MITO or HA-MITO constructs) were used to define flow cytometric gates. Illustrative plots are shown. On the left, the initial gate for single, viable cells (polygon) was set with 89.6% of events included for further analysis; the forward scatter (area) and side scatter (area) axes are linear. On the right, the second gate (i.e., for EGFP-positivity) was set with 99.92% of wildtype HeLa cells excluded and is shaded green. The light blue zone thus represents background EGFP signal. The forward scatter (area) axis is linear and the EGFP (area) axis is logarithmic. For each sample, 100,000 cells were analyzed on a FACSAria IIU SORP sorter (BD Biosciences) using the FACSDiva software (BD Biosciences) and the data was converted into contour plots with outliers using the FlowJo software (FlowJo). For this figure, HeLa cells were cultured in complete DMEM, authenticated by the Duke University DNA Analysis Facility, and tested for mycoplasma contamination.

Supplementary Figure 2 Microscopic features of cells, free nuclei, and free organelles in a homogenate.

Representative micrographs of a homogenate of HeLa cells expressing the HA-MITO construct. Cells were homogenized using 25 strokes without rotation of the plunger and a sample of the homogenate was diluted with KPBS and then transferred to the well of a 6 well plate for microscopy. The physical features (gray) and EGFP signals (green) of the components of the homogenate were assessed. TL, standard transmitted light microscopy; C, cells; N, free nuclei; O, free organelles. Scale bar, 10 μm. For this figure, HeLa cells were cultured in complete DMEM, authenticated by the Duke University DNA Analysis Facility, and tested for mycoplasma contamination.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2. (PDF 383 kb)

Supplementary Data 1

Comparison of matrix concentrations of mitochondrial metabolites using normal and lengthened isolation times (.xls file). (XLSX 59 kb)

Supplementary Data 2

List of internal standards used for normalizing various metabolites (.xls file). (XLSX 33 kb)

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Chen, W., Freinkman, E. & Sabatini, D. Rapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites. Nat Protoc 12, 2215–2231 (2017). https://doi.org/10.1038/nprot.2017.104

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