Abstract

We extended thermal proteome profiling to detect transmembrane protein–small molecule interactions in cultured human cells. When we assessed the effects of detergents on ATP-binding profiles, we observed shifts in denaturation temperature for ATP-binding transmembrane proteins. We also observed cellular thermal shifts in pervanadate-induced T cell–receptor signaling, delineating the membrane target CD45 and components of the downstream pathway, and with drugs affecting the transmembrane transporters ATP1A1 and MDR1.

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Acknowledgements

We thank J. Stuhlfauth for cell culture; M. Jundt, K. Kammerer, M. Klös-Hudak, M. Paulmann, I. Tögel and T. Rudi for expert technical assistance; and C.-W. Chung, J.W. Polli and M. Zamek-Gliszczynski for helpful suggestions.

Author information

Author notes

    • Friedrich B M Reinhard
    •  & Dirk Eberhard

    These authors contributed equally to this work.

Affiliations

  1. Cellzome GmbH, Molecular Discovery Research, GlaxoSmithKline, Heidelberg, Germany.

    • Friedrich B M Reinhard
    • , Dirk Eberhard
    • , Thilo Werner
    • , Holger Franken
    • , Dorothee Childs
    • , Carola Doce
    • , Maria Fälth Savitski
    • , Marcus Bantscheff
    • , Mikhail M Savitski
    •  & Gerard Drewes
  2. Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

    • Dorothee Childs
    •  & Wolfgang Huber

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Contributions

F.B.M.R., M.M.S. and G.D. conceived the project; F.B.M.R., D.E., T.W., M.B., M.M.S. and G.D. designed the experiments; F.B.M.R., D.E. and T.W. conducted and supervised experiments; F.B.M.R., T.W., H.F., D.C., M.F.S., C.D., W.H., M.B., M.M.S. and G.D. contributed to data analysis; F.B.M.R. and M.B. contributed to the manuscript; and M.M.S. and G.D. wrote the manuscript.

Competing interests

F.B.M.R., D.E., T.W., H.F., D.C., C.D., M.F.S., M.B., M.M.S. and G.D. are employees and/or shareholders of Cellzome GmbH and GlaxoSmithKline, companies that funded the work.

Corresponding authors

Correspondence to Mikhail M Savitski or Gerard Drewes.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7

Excel files

  1. 1.

    Supplementary Table 1

    Thermal proteome profiling data showing effect of ATP on proteins in lysate extracted in absence of detergent.

  2. 2.

    Supplementary Table 2

    Thermal proteome profiling data showing effect of ATP on proteins in lysate extracted in presence of detergent.

  3. 3.

    Supplementary Table 3

    Comparison of effect of ATP in detergent versus non-detergent conditions

  4. 4.

    Supplementary Table 4

    Thermal proteome profiling data obtained in intect cells showing effect of pervanadate on proteins and subsequent lysis in absence of detergent.

  5. 5.

    Supplementary Table 5

    Thermal proteome profiling data obtained in intect cells showing effect of pervanadate on proteins and subsequent lysis in presence of detergent.

  6. 6.

    Supplementary Table 6

    Comparison of effect of pervanadate on proteins in intact cells extracted post-heating in presence or absence of detergent.

  7. 7.

    Supplementary Table 7

    Pathway analysis on proteins shifted by pervanadate treatment using Ingenuity Pathway Analysis software.

  8. 8.

    Supplementary Table 8

    Effect of a concentration range of oubain to the thermal stability proteins at 63 °C

  9. 9.

    Supplementary Table 9

    Effect of a concentration range of elacridar to the thermal stability proteins at 54 °C and 56 °C

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DOI

https://doi.org/10.1038/nmeth.3652

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