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Identifying key membrane protein lipid interactions using mass spectrometry

Nature Protocols volume 13, pages 1106–1120 (2018)Cite this article

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Abstract

With the recent success in determining membrane protein structures, further detailed understanding of the identity and function of the bound lipidome is essential. Using an approach that combines high-energy native mass spectrometry (HE-nMS) and solution-phase lipid profiling, this protocol can be used to determine the identity of the endogenous lipids that directly interact with a protein. Furthermore, this method can identify systems in which such lipid binding has a major role in regulating the oligomeric assembly of membrane proteins. The protocol begins with recording of the native mass spectrum of the protein of interest, under successive delipidation conditions, to determine whether delipidation leads to disruption of the oligomeric state. Subsequently, we propose using a bipronged strategy: first, an HE-nMS platform is used that allows dissociation of the detergent micelle at the front end of the instrument. This allows for isolation of the protein–lipid complex at the quadrupole and successive fragmentation at the collision cell, which leads to identification of the bound lipid masses. Next, simultaneous coupling of this with in-solution LC-MS/MS-based identification of extracted lipids reveals the complete identity of the interacting lipidome that copurifies with the proteins. Assimilation of the results of these two sets of experiments divulges the complete identity of the set of lipids that directly interact with the membrane protein of interest, and can further delineate its role in maintaining the oligomeric state of the protein. The entire procedure takes 2 d to complete.

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Figure 1: Schematic representation of the processes involving the determination of bound lipids to membrane proteins.
Figure 2: Delipidation of dimeric LeuT: progressive delipidation of LeuT dimer in 1% (wt/vol) delipidating detergent NG.
Figure 3: Schematic of successive delipidation of MsbA.
Figure 4: Schematic of the high-energy platform and its application with various membrane proteins.
Figure 5: HE-nMS experiment on MATE.
Figure 6: HE-nMS of dimeric lipid-bound LeuT.
Figure 7: HE-nMS and in-solution lipid identification of semiSWEET.

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References

  1. Konijnenberg, A. et al. Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry. Proc. Natl. Acad. Sci. USA 111, 17170–17175 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Schiffrin, B. et al. Skp is a multivalent chaperone of outer-membrane proteins. Nat. Commun. 23, 786–793 (2016).

    CAS  Google Scholar 

  3. Harvey, S.R., Liu, Y., Liu, W., Wysocki, V.H. & Laganowsky, A. Surface induced dissociation as a tool to study membrane protein complexes. Chem. Commun. 53, 3106–3109 (2017).

    Article  CAS  Google Scholar 

  4. Zhang, H. et al. Native mass spectrometry characterizes the photosynthetic reaction center complex from the purple bacterium Rhodobacter sphaeroides. J. Am. Soc. Mass Spectrom. 28, 87–95 (2017).

    Article  PubMed  Google Scholar 

  5. Iadanza, M.G. et al. Lateral opening in the intact beta-barrel assembly machinery captured by cryo-EM. Nat. Commun. 7, 12865 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Landreh, M., Marklund, E.G., Uzdavinys, P. & Degiacomi, M.T. Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters. Nat. Commun. 8, 13993 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hedger, G. & Sansom, M.S. Lipid interaction sites on channels, transporters and receptors: recent insights from molecular dynamics simulations. Biochim. Biophys. Acta 1858, 2390–2400 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Landreh, M., Marty, M.T., Gault, J. & Robinson, C.V. A sliding selectivity scale for lipid binding to membrane proteins. Curr. Opin. Struct. Biol. 39, 54–60 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Norimatsu, Y., Hasegawa, K., Shimizu, N. & Toyoshima, C. Protein-phospholipid interplay revealed with crystals of a calcium pump. Nature 545, 193–198 (2017).

    Article  CAS  PubMed  Google Scholar 

  11. Le Roy, A. et al. AUC and small-angle scattering for membrane proteins. Methods Enzymol 562, 257–286 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Maekawa, S., Kobayashi, Y., Morita, M. & Suzaki, T. Tight binding of NAP-22 with acidic membrane lipids. Neurosci. Lett. 600, 244–248 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Deme, J.C. et al. Purification and interaction analyses of two human lysosomal vitamin B12 transporters: LMBD1 and ABCD4. Mol. Membr. Biol. 31, 250–261 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Zhou, M. et al. Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding. Science 334, 380–385 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bechara, C. et al. A subset of annular lipids is linked to the flippase activity of an ABC transporter. Nat. Chem. 7, 255–262 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Gault, J. et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Marcoux, J. et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl. Acad. Sci. USA 110, 9704–9709 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Liko, I. et al. Dimer interface of bovine cytochrome c oxidase is influenced by local posttranslational modifications and lipid binding. Proc. Natl. Acad. Sci. USA 113, 8230–8235 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mehmood, S. et al. Mass spectrometry captures off-target drug binding and provides mechanistic insights into the human metalloprotease ZMPSTE24. Nat. Chem. 8, 1152–1158 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Laganowsky, A., Reading, E., Hopper, J.T. & Robinson, C.V. Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639–651 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sun, J., Kitova, E.N., Sun, N. & Klassen, J.S. Method for identifying nonspecific protein-protein interactions in nanoelectrospray ionization mass spectrometry. Anal. Chem. 79, 8301–8311 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Ilgu, H. et al. Variation of the detergent-binding capacity and phospholipid content of membrane proteins when purified in different detergents. Biophys. J. 106, 1660–1670 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sun, J., Kitova, E.N. & Klassen, J.S. Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry. Anal. Chem. 79, 416–425 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Campuzano, I.D. et al. Native MS analysis of bacteriorhodopsin and an empty nanodisc by orthogonal acceleration time-of-flight, orbitrap and ion cyclotron resonance. Anal. Chem. 88, 12427–12436 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Barrera, N.P., Di Bartolo, N., Booth, P.J. & Robinson, C.V. Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Liko, I., Hopper, J.T., Allison, T.M., Benesch, J.L. & Robinson, C.V. Negative ions enhance survival of membrane protein complexes. J. Am. Soc. Mass Spectrom. 27, 1099–1104 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hernandez, H. & Robinson, C.V. Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat. Protoc. 2, 715–726 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Bird, S.S., Marur, V.R., Sniatynski, M.J., Greenberg, H.K. & Kristal, B.S. Lipidomics profiling by high-resolution LC-MS and high-energy collisional dissociation fragmentation: focus on characterization of mitochondrial cardiolipins and monolysocardiolipins. Anal. Chem. 83, 940–949 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Allison, T.M. et al. Quantifying the stabilizing effects of protein-ligand interactions in the gas phase. Nat. Commun. 6, 8551 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Mehmood, S. et al. Charge reduction stabilizes intact membrane protein complexes for mass spectrometry. J. Am. Chem. Soc. 136, 17010–17012 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. http://www.lipidmaps.org/data/structure/index.php.

  32. Tsugawa, H. et al. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat. Methods 12, 523–526 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The Robinson group is funded by a Wellcome Trust Investigator Award (104633/Z/14/Z), an ERC Advanced Grant ENABLE (641317) and an MRC program grant (MR/N020413/1). K.G. is a Junior Research Fellow at St Catherine's College, Oxford, and is supported by the Royal Commission for the Exhibition of 1851. J.G. is a Junior Research Fellow of The Queen's College. J. Donlan, Waters Corporation and OMass Technologies are thanked for their support.

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  1. Idlir Liko & Jonathan T S Hopper

    Present address: Present address: OMass Technologies Ltd, Centre for Innovation & Enterprise, Oxford, UK.,

Authors and Affiliations

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

    Kallol Gupta, Jingwen Li, Idlir Liko, Joseph Gault, Cherine Bechara, Di Wu, Jonathan T S Hopper, Justin L P Benesch & Carol V Robinson

  2. Institut de Genomique Fonctionnelle, CNRS UMR-5203, INSERM U1191, University of Montpellier, Montpellier, France.,

    Cherine Bechara

  3. Waters Corporation, Wilmslow, UK

    Kevin Giles

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Contributions

K. Gupta expressed and purified LeuT and SemiSWEET, and performed the high-energy experiments with the help of J.T.S.H. C.B. developed the successive delipidation protocol, and J.L. expressed and purified MsbA and performed the successive delipidation experiment. C.B. and J.G. performed the lipidomics experiments and analyzed the data. D.W. set up the lipidomics platform. I.L. expressed, purified and performed the MS experiments on MATE. J.L.P.B., J.T.S.H. and K. Giles designed the high-energy source. K. Gupta and C.V.R. wrote the manuscript with assistance from J.G. and I.L. and input from all other authors.

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Correspondence to Carol V Robinson.

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Gupta, K., Li, J., Liko, I. et al. Identifying key membrane protein lipid interactions using mass spectrometry. Nat Protoc 13, 1106–1120 (2018). https://doi.org/10.1038/nprot.2018.014

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