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Mass Spectrometry–Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential iodoTMT Labeling

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Book cover Plant Mitochondria

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2363))

Abstract

Mitochondria are central hubs of redox biochemistry in the cell. An important role of mitochondrial carbon metabolism is to oxidize respiratory substrates and to pass the electrons down the mitochondrial electron transport chain to reduce oxygen and to drive oxidative phosphorylation. During respiration, reactive oxygen species are produced as a side reaction, some of which in turn oxidize cysteine thiols in proteins. Hence, the redox status of cysteine-containing mitochondrial proteins has to be controlled by the mitochondrial glutathione and thioredoxin systems, which draw electrons from metabolically derived NADPH. The redox status of mitochondrial cysteines can undergo fast transitions depending on the metabolic status of the cell, as for instance at early seed germination. Here, we describe a state-of-the-art method to quantify redox state of protein cysteines in isolated Arabidopsis seedling mitochondria of controlled metabolic and respiratory state by MS2-based redox proteomics using the isobaric thiol labeling reagent Iodoacetyl Tandem Mass Tag™ (iodoTMT). The procedure is also applicable to isolated mitochondria of other plant and nonplant systems.

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Acknowledgments

We would like to thank Thomas Nietzel and Falko Hochgräfe for their previous work on the iodoTMT method development, on which aspects of the presented method were optimized.

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Authors and Affiliations

  1. Institute of Plant Biology and Biotechnology, University of Münster, Münster, Germany

    Jonas Giese, Jürgen Eirich, Frederik Post, Markus Schwarzländer & Iris Finkemeier

Authors
  1. Jonas Giese
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  2. Jürgen Eirich
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  3. Frederik Post
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  4. Markus Schwarzländer
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  5. Iris Finkemeier
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Corresponding author

Correspondence to Iris Finkemeier .

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Editors and Affiliations

  1. Department of Biology, Lund University, Lund, Sweden

    Olivier Van Aken

  2. Department of Biology, Lund University, Lund, Sweden

    Allan G. Rasmusson

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Giese, J., Eirich, J., Post, F., Schwarzländer, M., Finkemeier, I. (2022). Mass Spectrometry–Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential iodoTMT Labeling. In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_16

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  • DOI: https://doi.org/10.1007/978-1-0716-1653-6_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1652-9

  • Online ISBN: 978-1-0716-1653-6

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