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Identifying key membrane protein lipid interactions using mass spectrometry

Nature Protocols volume 13, pages 11061120 (2018) | Download Citation


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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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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Author notes

    • Idlir Liko
    •  & Jonathan T S Hopper

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


  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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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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The authors declare no competing financial interests.

Corresponding author

Correspondence to Carol V Robinson.

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