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Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation

Nature Protocols volume 16, pages 5083–5122 (2021)Cite this article

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Abstract

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry–based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional ‘capture reagents’ by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease.

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Fig. 1: Background: types of lipid PTMs covered and the corresponding lipid probes.
Fig. 2: Schematic overview of the procedures presented.
Fig. 3: Procedure 1: in-gel fluorescence visualization of lipid probe–labeled proteins.
Fig. 4: Procedure 2: identification and quantification of lipid PTMs by chemical proteomics.
Fig. 5: Anticipated results: visualization of protein lipidation by using the presented lipid probes.
Fig. 6: Anticipated results: visualizing target engagement of lipid transferases via metabolic labeling with lipid probes.
Fig. 7: Anticipated results: identifying and quantifying target engagement of lipid transferase inhibitors by using chemical proteomics.

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Data availability

The gel lanes depicted in Figs. 5 and 6 are shown uncropped in Supplementary Figs. 1 and 2. The MS proteomics data used for Fig. 7 have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022490.

Code availability

MaxQuant and Perseus are freely available from https://www.maxquant.org.

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Acknowledgements

The authors thank L. Haig for assistance with MS, R. Serwa for aiding in setup of the proteomics protocols and R. Singh for proofreading. The Francis Crick Institute Cell Services are acknowledged for providing cell lines and STR verification. This work was supported by grants from The Royal Society (Newton International Fellowship grant NF161582 to W.W.K.), the European Commission (Marie Sklodowska Curie Individual Fellowship grant 752165 to W.W.K. and Marie Sklodowska Curie Intra European Fellowship to J.M.-S. and E.W.T.), the Royal Thai Government scholarship (PhD studentship to N.P.), the Imperial College London Institute of Chemical Biology EPSRC Centre for Doctoral Training (grant EP/F500416/1 to P.C.), EPSRC (Impact Acceleration Account grant PS1042 to W.W.K. and E.W.T.) and Cancer Research UK (C29637/A21451 and C29637/A20183 to E.W.T.). Work in the E.W.T. laboratories is supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001057 and FC001097), the UK Medical Research Council (FC001057 and FC001097) and the Wellcome Trust (FC001057 and FC001097).

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Author notes

  1. Thomas Lanyon-Hogg

    Present address: Department of Pharmacology, University of Oxford, Oxford, UK

  2. Nattawadee Panyain

    Present address: Global Health Institute, Faculty of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland

  3. Paulina Ciepla

    Present address: Institute of Chemical Sciences and Engineering (ISIC), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland

  4. Julia Morales-Sanfrutos

    Present address: Proteomics Unit, Biotechnology Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain

Authors and Affiliations

  1. Department of Chemistry, Imperial College London, Molecular Sciences Research Hub, London, UK

    Wouter W. Kallemeijn, Thomas Lanyon-Hogg, Nattawadee Panyain, Andrea Goya Grocin, Paulina Ciepla, Julia Morales-Sanfrutos & Edward W. Tate

  2. The Francis Crick Institute, London, UK

    Wouter W. Kallemeijn, Andrea Goya Grocin & Edward W. Tate

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Contributions

W.W.K., N.P., T.L.-H. and E.W.T. were responsible for conceptualization. W.W.K., N.P., A.G.G. and J.M.-S. were responsible for methodology. W.W.K., T.L.H., N.P., A.G.G., J.M.-S. and P.C. were responsible for investigation. E.W.T. was responsible for resources. W.W.K. and T.L.H. wrote the original draft. All authors reviewed and edited the manuscript. W.W.K. was responsible for visualization. E.W.T. provided supervision.

Corresponding author

Correspondence to Edward W. Tate.

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Competing interests

E.W.T. is a director and shareholder of Myricx Pharma Ltd. and an inventor on a patent application describing NMT inhibitors including IMP-1088 (Bell, A. S., Tate, E. W., Leatherbarrow, R. J., Hutton, J. A., Brannigan, J. A. Compounds and their use as inhibitors of N-myristoyl transferase. PCT In Appl. (2017) WO 2017001812).

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Peer review information Nature Protocols thanks Matthew Pratt, Tamara Kinzer-Ursem and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Kallemeijn, W. W. et al. Cell Chem. Biol. 26, 892–900.e4 (2019): https://doi.org/10.1016/j.chembiol.2019.03.006

Storck, E. M. et al. Nat. Chem. 11, 552–561 (2019): https://doi.org/10.1038/s41557-019-0237-6

Mousnier, A. et al. Nat. Chem. 10, 599–606 (2018): https://doi.org/10.1038/s41557-018-0039-2

Broncel, M. et al. Angew. Chem. Int. Ed. Engl. 54, 5948–5951 (2015): https://doi.org/10.1002/anie.201500342

Thinon, E. et al. Nat. Commun. 5, 4919 (2014): https://doi.org/10.1038/ncomms5919

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Kallemeijn, W.W., Lanyon-Hogg, T., Panyain, N. et al. Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation. Nat Protoc 16, 5083–5122 (2021). https://doi.org/10.1038/s41596-021-00601-6

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