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Cell surface thermal proteome profiling tracks perturbations and drug targets on the plasma membrane

Nature Methods volume 18, pages 84–91 (2021)Cite this article

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Abstract

Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug.

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Fig. 1: CS-TPP enables selective monitoring of thermal stability and abundance changes of plasma membrane proteins.
Fig. 2: Target engagement to receptor kinases.
Fig. 3: Monitoring T cell activation by CS-TPP.
Fig. 4: Targeting integrins and heat shock proteins.
Fig. 5: Target engagement to the G-protein-coupled receptor CXCR4.

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Data availability

All source data are available in the main text or the supplementary materials. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE59 partner repository with the dataset identifier PXD016249. Annotations of proteins were based on the UniProt database (14 December 2016, https://www.uniprot.org/).

Code availability

An implementation of the above described CS-TPP analysis procedure can be found at https://github.com/mathiaskalxdorf/RTSA or at https://doi.org/10.5281/zenodo.4274152.

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Acknowledgements

We thank J. Stuhlfauth, N. Garcia-Altrieth, K. Beß and B. Dlugosch for the supporting cell culture production; M. Bösche, T. Rudi, M. Klös-Hudak, K. Kammerer and M. Steidel for assistance with mass spectrometry and C. Boecker and T. Mathieson for the IT and computational support.

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Authors and Affiliations

  1. Cellzome GmbH, GlaxoSmithKline (GSK), Heidelberg, Germany

    Mathias Kalxdorf, Ina Günthner, Sascha Knecht, H. Christian Eberl & Marcus Bantscheff

  2. Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Isabelle Becher, Nils Kurzawa & Mikhail M. Savitski

  3. Faculty of Biosciences, Joint PhD Degree Program from EMBL and Heidelberg University, Heidelberg, Germany

    Nils Kurzawa

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Contributions

M.K. and H.C.E. designed the experiments. M.K. and I.G. performed the CS-TPP experiments. I.B. and S.K. performed lactate assay experiments. M.K. analyzed the CS-TPP data. N.K performed TPCA analysis. I.B. reviewed the figures. M.M.S. gave scientific advice. M.K., H.C.E. and M.B. wrote the manuscript. M.B. and H.C.E. supervised the work.

Corresponding authors

Correspondence to H. Christian Eberl or Marcus Bantscheff.

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Competing interests

M.K., I.G., S.K., H.C.E. and M.B. are GSK employees. M.M.S. is a GSK shareholder.

Additional information

Peer review information Arunima, Singh was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8.

Reporting Summary

Supplementary Table 1

Source data for the comparison of conventional TPP and TPP with cell surface enrichment on the example of cellular treatment with 1 µM ouabain.

Supplementary Table 2

Source data for all CS-TPP experiments.

Supplementary Table 3

Source data the gene ontology enrichment analyses comparing proteins with substantially different melting points (ΔTM > ±4 °C) between conventional TPP and TPP with additional cell surface enrichment.

Supplementary Table 4

Source data for the correlation of thermal stability with protein properties.

Supplementary Table 5

Summary of information about additionally affected proteins in CS-TPP experiments.

Supplementary Table 6

Source data for the TPCA analysis.

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Kalxdorf, M., Günthner, I., Becher, I. et al. Cell surface thermal proteome profiling tracks perturbations and drug targets on the plasma membrane. Nat Methods 18, 84–91 (2021). https://doi.org/10.1038/s41592-020-01022-1

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