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Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters

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Abstract

The mammalian membrane is composed of various eukaryotic lipids interacting with extensively post-translationally modified proteins. Probing interactions between these mammalian membrane proteins and their diverse and heterogeneous lipid cohort remains challenging. Recently, native mass spectrometry (MS) combined with bottom-up ‘omics’ approaches has provided valuable information to relate structural and functional lipids to membrane protein assemblies in eukaryotic membranes. Here we provide a step-by-step protocol to identify and provide relative quantification for endogenous lipids bound to mammalian membrane proteins and their complexes. Using native MS to guide our lipidomics strategies, we describe the necessary sample preparation steps, followed by native MS data acquisition, tailored lipidomics and data interpretation. We also highlight considerations for the integration of different levels of information from native MS and lipidomics and how to deal with the various challenges that arise during the experiments. This protocol begins with the preparation of membrane proteins from mammalian cells and tissues for native MS. The results enable not only direct assessment of copurified endogenous lipids but also determination of the apparent affinities of specific lipids. Detailed sample preparation for lipidomics analysis is also covered, along with comprehensive settings for liquid chromatography–MS analysis. This protocol is suitable for the identification and quantification of endogenous lipids, including fatty acids, sterols, glycerolipids, phospholipids and glycolipids and can be used to interrogate proteins from recombinant sources to native membranes.

Key points

  • Lipids have important structural and functional roles in membrane protein assemblies, for example, receptors and transporters. However, it is challenging to get precise structural information, especially when the assemblies are large and the proteins have extensive posttranslational modifications.

  • Native mass spectrometry is a top-down approach that can be adapted to handle this increase in complexity. This protocol describes how native mass spectrometry can be combined with bottom-up lipidomic analysis.

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Fig. 1: Overview of the protocol workflow.
Fig. 2: The most common lipids detected in MS-based lipidomics.
Fig. 3: MS analysis of SPNS2–lipid interactions.
Fig. 4: The isotopic envelope of a protein complex limits the resolution of bound lipids due to the heterogeneity of PTMs on the protein complexes.
Fig. 5: MS analysis of the LAT1–4F2hc complex.
Fig. 6: MS analysis of GPR158 dimer.
Fig. 7: MS analysis of mammalian V-type ATPase.
Fig. 8: MS analysis of bovine rhodopsin in lipid vesicles formed from bovine rod disc membranes.

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Data availability

Raw native MS data of bovine rhodopsin are deposited in figshare (https://doi.org/10.6084/m9.figshare.25391746). All other data are available from the corresponding author upon reasonable request.

References

  1. Levental, I. & Lyman, E. Regulation of membrane protein structure and function by their lipid nano-environment. Nat. Rev. Mol. Cell Biol. 24, 107–122 (2023).

    Article  CAS  PubMed  Google Scholar 

  2. Corradi, V. et al. Emerging diversity in lipid–protein interactions. Chem. Rev. 119, 5775–5848 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Leney, A. C. & Heck, A. J. R. Native mass spectrometry: what is in the name? J. Am. Soc. Mass Spectrom. 28, 5–13 (2017).

    Article  CAS  PubMed  Google Scholar 

  4. Bolla, J. R., Agasid, M. T., Mehmood, S. & Robinson, C. V. Membrane protein–lipid interactions probed using mass spectrometry. Annu. Rev. Biochem. 88, 85–111 (2019).

    Article  CAS  PubMed  Google Scholar 

  5. Gupta, K. et al. Identifying key membrane protein lipid interactions using mass spectrometry. Nat. Protoc. 13, 1106–1120 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yen, H.-Y. et al. PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature 559, 423–427 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Chorev, D. S. et al. Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry. Science 362, 829–834 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bolla, J. R. et al. Direct observation of the influence of cardiolipin and antibiotics on lipid II binding to MurJ. Nat. Chem. 10, 363–371 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Miliara, X. et al. Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins. Nat. Commun. 10, 1130 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  10. McDowell, M. A. et al. Structural basis of tail-anchored membrane protein biogenesis by the GET insertase complex. Mol. Cell 80, 72–86.e7 (2020).

    Article  CAS  PubMed  Google Scholar 

  11. Jin, R. et al. Ion currents through Kir potassium channels are gated by anionic lipids. Nat. Commun. 13, 490 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Schmidpeter, P. A. M. et al. Anionic lipids unlock the gates of select ion channels in the pacemaker family. Nat. Struct. Mol. Biol. 121, 438a–439a (2022).

    Google Scholar 

  13. Yao, X. et al. Structures of the R-type human Cav2.3 channel reveal conformational crosstalk of the intracellular segments. Nat. Commun. 13, 7358 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Patil, D. N. et al. Cryo-EM structure of human GPR158 receptor coupled to the RGS7–Gβ5 signaling complex. Science 375, 86–91 (2022).

    Article  CAS  PubMed  Google Scholar 

  15. Sobti, M. et al. Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates symmetry mismatch. Nat. Commun. 11, 2615 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vasanthakumar, T. et al. Structural comparison of the vacuolar and Golgi V-ATPases from Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 116, 7272–7277 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Abbas, Y. M., Wu, D., Bueler, S. A., Robinson, C. V. & Rubinstein, J. L. Structure of V-ATPase from the mammalian brain. Science 367, 1240–1246 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wang, L., Wu, D., Robinson, C. V., Wu, H. & Fu, T. Structures of a complete human V-ATPase reveal mechanisms of its assembly. Mol. Cell 80, 501–511.e3 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wu, D. et al. The complete assembly of human LAT1–4F2hc complex provides insights into its regulation, function and localisation. Nat. Commun. 15, 3711 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tang, H. et al. The solute carrier SPNS2 recruits PI(4,5)P2 to synergistically regulate transport of sphingosine-1-phosphate. Mol. Cell 83, 2739–2752.e5 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, S. et al. Capturing a rhodopsin receptor signalling cascade across a native membrane. Nature 604, 384–390 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 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 

  24. Gault, J. et al. Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins. Nat. Methods 17, 505–508 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 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 

  26. Kostelic, M. M., Ryan, A. M., Reid, D. J., Noun, J. M. & Marty, M. T. Expanding the types of lipids amenable to native mass spectrometry of lipoprotein complexes. J. Am. Soc. Mass Spectrom. 30, 1416–1425 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Dunham, W. H., Mullin, M. & Gingras, A.-C. Affinity-purification coupled to mass spectrometry: basic principles and strategies. Proteomics 12, 1576–1590 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Biou, V. Lipid-membrane protein interaction visualised by cryo-EM: a review. Biochim. Biophys. Acta Biomembr. 1865, 184068 (2023).

    Article  CAS  PubMed  Google Scholar 

  29. Bagheri, Y., Ali, A. A. & You, M. Current methods for detecting cell membrane transient interactions. Front. Chem. 8, 1–14 (2020).

    Article  Google Scholar 

  30. Wang, L., Wu, D., Robinson, C. V. & Fu, T.-M. Identification of mEAK-7 as a human V-ATPase regulator via cryo-EM data mining. Proc. Natl Acad. Sci. USA 119, 1–3 (2022).

    CAS  Google Scholar 

  31. Shin, J. et al. Constitutive activation mechanism of a class C GPCR. Nat. Struct. Mol. Biol. 31, 678–687 (2024).

    Article  CAS  PubMed  Google Scholar 

  32. Oluwole, A. O. et al. Peptidoglycan biosynthesis is driven by lipid transfer along enzyme-substrate affinity gradients. Nat. Commun. 13, 2278 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fiorentino, F. et al. Dynamics of an LPS translocon induced by substrate and an antimicrobial peptide. Nat. Chem. Biol. 17, 187–195 (2021).

    Article  CAS  PubMed  Google Scholar 

  34. Wenk, M. R. et al. Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nat. Biotechnol. 21, 813–817 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Yan, R., Zhao, X., Lei, J. & Zhou, Q. Structure of the human LAT1–4F2hc heteromeric amino acid transporter complex. Nature 568, 127–130 (2019).

    Article  CAS  PubMed  Google Scholar 

  36. 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 

  37. Lutomski, C. A. et al. Infrared multiphoton dissociation enables top-down characterization of membrane protein complexes and G protein-coupled receptors. Angew. Chem. Int. Ed. Engl. 62, e202305694 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Durbin, K. R. et al. ProSight native: defining protein complex composition from native top-down mass spectrometry data. J. Proteome Res. 22, 2660–2668 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Mkam Tsengam, I. K. et al. Transformation of lipid vesicles into micelles by adding nonionic surfactants: elucidating the structural pathway and the intermediate structures. J. Phys. Chem. B 126, 2208–2216 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hutchins, P. D., Russell, J. D. & Coon, J. J. LipiDex: an integrated software package for high-confidence lipid identification. Cell Syst. 6, 621–625.e5 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Pluskal, T., Castillo, S., Villar-Briones, A. & Orešič, M. MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinform. 11, 395 (2010).

    Article  Google Scholar 

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

    Article  PubMed  Google Scholar 

  44. Cong, X. et al. Determining membrane protein–lipid binding thermodynamics using native mass spectrometry. J. Am. Chem. Soc. 138, 4346–4349 (2016).

    Article  CAS  PubMed  Google Scholar 

  45. Schrecke, S. et al. Selective regulation of human TRAAK channels by biologically active phospholipids. Nat. Chem. Biol. 17, 89–95 (2021).

    Article  CAS  PubMed  Google Scholar 

  46. Matyash, V., Liebisch, G., Kurzchalia, T. V., Shevchenko, A. & Schwudke, D. Lipid extraction by methyl-terf-butyl ether for high-throughput lipidomics. J. Lipid Res. 49, 1137–1146 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chambers, M. C. et al. A cross-platform toolkit for mass spectrometry and proteomics. Nat. Biotechnol. 30, 918–920 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

D.W., H.T., X.Q., S.C. and C.V.R. are grateful for the support of the Wellcome Trust grant number 221795/Z/20/Z. S.S. is funded by CSC–University of Oxford Scholarship. To provide open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

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Author notes

  1. These authors contributed equally: Di Wu, Haiping Tang, Xingyu Qiu.

Authors and Affiliations

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

    Di Wu, Haiping Tang, Xingyu Qiu, Siyuan Song, Siyun Chen & Carol V. Robinson

  2. Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, UK

    Di Wu, Haiping Tang, Xingyu Qiu, Siyuan Song, Siyun Chen & Carol V. Robinson

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  1. Di Wu
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Contributions

D.W. performed MS analysis of LAT1–4F2hc and V-type ATPase. H.T. performed MS analysis of SPNS2. X.Q. and D.W. performed MS analysis of GPR158, S.C., S.S., X.Q. and D.W. performed MS analysis of rhodopsin. D.W., H.T., X.Q. and C.V.R. wrote the manuscript with input from all other authors.

Corresponding author

Correspondence to Carol V. Robinson.

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Competing interests

C.V.R. is a cofounder and consultant of OMass Therapeutics. The other authors declare no competing interests.

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Related links

Key references using this protocol:

Wu, D. et al. Nat. Commun. 15, 3711 (2024): https://doi.org/10.1038/s41467-024-47948-4

Tang, H. et al. Mol. Cell 83, 2739–2752.e5 (2023): https://doi.org/10.1016/j.molcel.2023.06.033

Chen, S. et al. Nature 604, 384–390 (2022): https://doi.org/10.1038/s41586-022-04547-x

Wang, L. et al. Mol. Cell 80, 501–511.e3 (2020): https://doi.org/10.1016/j.molcel.2020.09.029

Patil, D. N. et al. Science 375, 86–91 (2022): https://doi.org/10.1126/science.abl4732

This protocol is an update to: Nat. Protoc. 13, 1106–1120 (2018): https://doi.org/10.1038/nprot.2018.014

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Wu, D., Tang, H., Qiu, X. et al. Native MS-guided lipidomics to define endogenous lipid microenvironments of eukaryotic receptors and transporters. Nat Protoc (2024). https://doi.org/10.1038/s41596-024-01037-4

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