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Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions

Nature Methods volume 6, pages 585–587 (2009)Cite this article

Abstract

We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.

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Figure 1: Subunit stoichiometry and lipid binding for two ABC transporters.
Figure 2: Incorporation of modified subunits in the EmrE dimer.
Figure 3: Trimers of MexB enable homology modeling.

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Acknowledgements

We acknowledge funding from the Biotechnology and Biological Sciences Research Council, Medical Research Council, European Molecular Biology Organization, Swiss National Science Foundation, The Wellcome Trust, Royal Society, European Union PROSPECTS and Walters-Kundert Trust. We thank M. Welch (University of Cambridge) for P. aeruginosa genomic DNA.

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Authors and Affiliations

  1. Department of Chemistry, University of Cambridge, Cambridge, UK

    Nelson P Barrera, Shoshanna C Isaacson, Min Zhou & Carol V Robinson

  2. Department of Physics, University of Oxford, Clarendon Laboratory, Oxford, UK

    Vassiliy N Bavro

  3. Department of Pharmacology, University of Cambridge, Cambridge, UK

    Alex Welch, Theresia A Schaedler, Markus A Seeger, Hendrik W van Veen & Henrietta Venter

  4. Department of Biochemistry, University of Cambridge, Cambridge, UK

    Ricardo Núñez Miguel

  5. Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Vladimir M Korkhov & Christopher G Tate

  6. School of Biological and Biomedical Sciences, Durham University, Durham, UK

    Adrian R Walmsley

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  1. Nelson P Barrera
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Correspondence to Carol V Robinson.

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Supplementary Figures 1–4, Supplementary Tables 1–3 and Supplementary Protocol (PDF 535 kb)

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Barrera, N., Isaacson, S., Zhou, M. et al. Mass spectrometry of membrane transporters reveals subunit stoichiometry and interactions. Nat Methods 6, 585–587 (2009). https://doi.org/10.1038/nmeth.1347

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