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

Nature Protocols volume 20, pages 1–25 (2025)Cite this article

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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.

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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 20, 1–25 (2025). https://doi.org/10.1038/s41596-024-01037-4

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