Abstract
Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.
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Abbreviations
- DC:
-
differential centrifugation
- DC+MACS:
-
magnetic separation of crude mitochondria fraction obtained by DC
- MACS:
-
magnetic separation performed in full compliance with the Miltenyi Biotec protocol
- Sonication+DC+ MACS:
-
modification of the DC+MACS protocol in which homogenization in hypoosmotic buffer was replaced by sonication in sucrose buffer
- WCL:
-
whole cell lysate
References
Nunnari, J., and Suomalainen, A. (2012) Mitochondria: in sickness and in health, Cell, 148, 1145–1159.
Zong, W. X., Rabinowitz, J. D., and White, E. (2016) Mitochondria and cancer, Mol. Cell, 61, 667–676.
D’Aquila, P., Bellizzi, D., and Passarino, G. (2015) Mitochondria in health, aging and diseases: the epigenetic perspective, Biogerontology, 16, 569–585.
Palmfeldt, J., and Bross, P. (2017) Proteomics of human mitochondria, Mitochondrion, 33, 2–14.
Picard, M., Wallace, D. C., and Burelle, Y. (2016) The rise of mitochondria in medicine, Mitochondrion, 30, 105–116.
Eremina, L., Pashintseva, N., Kovalev, L., Kovaleva, M., and Shishkin, S. (2017) Proteomics of mammalian mitochondria in health and malignancy: from protein identification to function, Anal. Biochem., doi: 10.1016/j.ab. 2017.03.024.
Witter, R. F., Watson, M. L., and Cottone, M. A. (1955) Morphology and ATPase of isolated mitochondria, J. Biophys. Biochem. Cytol., 1, 127–138.
Weinbach, E. C. (1961) A procedure for isolating stable mitochondria from rat liver and kidney, Anal. Biochem., 2, 335–343.
Picard, M., Taivassalo, T., Ritchie, D., Wright, K. J., Thomas, M. M., Romestaing, C., and Hepple, R. T. (2011) Mitochondrial structure and function are disrupted by standard isolation methods, PLoS One, 6, e18317.
Kappler, L., Li J., Haring, H. U., Weigert, C., Lehmann, R., Xu, G., and Hoene, M. (2016) Purity matters: a workflow for the valid high-resolution lipid profiling of mitochondria from cell culture samples, Sci. Rep., 6, 21107.
Hornig-Do, H. T., Gunther, G., Bust, M., Lehnartz, P., Bosio, A., and Wiesner, R. J. (2009) Isolation of functional pure mitochondria by superparamagnetic microbeads, Anal. Biochem., 389, 1–5.
Rabilloud, T. (2008) Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis, Methods Mol. Biol., 432, 83–100.
Afanasyeva, M. A., Britanova, L. V., Korneev, K. V., Mitkin, N. A., Kuchmiy, A. A., and Kuprash, D. V. (2014) Clusterin is a potential lymphotoxin beta receptor target that is upregulated and accumulates in germinal centers of mouse spleen during immune response, PLoS One, 9, e98349.
Brechalov, A. V., Georgieva, S. G., and Soshnikova, N. V. (2014) Mammalian cells contain two functionally distinct PBAF complexes incorporating different isoforms of PHF10 signature subunit, Cell Cycle, 13, 1970–1979.
Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., and Pinton, P. (2009) Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells, Nat. Protoc., 4, 1582–1590.
Chaiyarit, S., and Thongboonkerd, V. (2009) Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study, Anal. Biochem., 394, 249–258.
Geiger, T., Wehner, A., Schaab, C., Cox, J., and Mann, M. (2012) Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins, Mol. Cell. Proteomics, 11, M111.014050.
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Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-461, November 27, 2017.
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Afanasyeva, M.A., Ustiugova, A.S., Golyshev, S.A. et al. Isolation of large amounts of highly pure mitochondria for “omics” studies. Biochemistry Moscow 83, 76–85 (2018). https://doi.org/10.1134/S0006297918010108
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DOI: https://doi.org/10.1134/S0006297918010108