Abstract

We develop an approach to tag proteomes synthesized by specific cell types in dissociated cortex, brain slices, and the brains of live mice. By viral-mediated expression of an orthogonal pyrrolysyl-tRNA synthetase-tRNAXXX pair in a cell type of interest and providing a non-canonical amino acid with a chemical handle, we selectively label neuronal or glial proteomes. The method enables the identification of proteins from spatially and genetically defined regions of the brain.

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References

  1. 1.

    , , & Nat. Neurosci. 17, 1491–1499 (2014).

  2. 2.

    , , , & Nat. Neurosci. 19, 1131–1141 (2016).

  3. 3.

    et al. Nat. Neurosci. 18, 1819–1831 (2015).

  4. 4.

    et al. Neuron 86, 1000–1014 (2015).

  5. 5.

    , , , & Cell Chem. Biol. 23, 805–815 (2016).

  6. 6.

    et al. Nat. Biotechnol. 32, 465–472 (2014).

  7. 7.

    et al. J. Am. Chem. Soc. 131, 8720–8721 (2009).

  8. 8.

    et al. Nat. Chem. Biol. 12, 776–778 (2016).

  9. 9.

    , & Gene Ther. 10, 337–347 (2003).

  10. 10.

    et al. J. Neurol. Sci. 35, 147–155 (1978).

  11. 11.

    , , & Brain Res. 200, 13–21 (1980).

  12. 12.

    , , , & Neuron 93, 1420–1435.e5 (2017).

  13. 13.

    et al. Nat. Neurosci. 13, 133–140 (2010).

  14. 14.

    , , & J. Neurosci. 28, 7025–7030 (2008).

  15. 15.

    et al. Nat. Biotechnol. 21, 562–565 (2003).

  16. 16.

    Annu. Rev. Neurosci. 32, 127–147 (2009).

  17. 17.

    , & J. Am. Chem. Soc. 132, 3640–3641 (2010).

  18. 18.

    , , , & Chem. Biol. 17, 1212–1222 (2010).

  19. 19.

    et al. Nat. Biotechnol. 35, 1196–1201 (2017).

  20. 20.

    Nature 550, 53–60 (2017).

  21. 21.

    & Nat. Biotechnol. 26, 1367–1372 (2008).

  22. 22.

    et al. J. Proteome Res. 10, 1794–1805 (2011).

  23. 23.

    et al. Nat. Methods 13, 731–740 (2016).

  24. 24.

    , & Nat. Protoc. 4, 44–57 (2009).

  25. 25.

    et al. Proc. Natl. Acad. Sci. 104, 16438–16443 (2007).

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Acknowledgements

This work was supported by the Medical Research Council, UK (MC_U105181009 and MC_UP_A024_1008, to J.W.C., MC_U105170643 to M.H.) V.B. is supported by an MRC case studentship (Nikon). M.G.S. and L.C. acknowledge the support of the NC3Rs, ARUK and Cure PD. We are grateful to K. Heesom for mass spectrometry, and the MRC-LMB Mass Spectrometry Facility for additional mass spectrometry. We thank H. Bagci for contributions to the synthesis of AlkK, F. Morgese for assistance with AAV preparations, and the MRC biomedical staff at ARES and the Cambridge University Biomedical staff at Phenomics for their help.

Author information

Author notes

    • Toke P Krogager
    • , Russell J Ernst
    •  & Thomas S Elliott

    These authors contributed equally to this work.

Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, England, UK.

    • Toke P Krogager
    • , Russell J Ernst
    • , Thomas S Elliott
    • , Václav Beránek
    • , Ernesto Ciabatti
    • , Marco Tripodi
    • , Michael H Hastings
    •  & Jason W Chin
  2. Department of Clinical Neurosciences, University of Cambridge, Cambridge, England, UK.

    • Laura Calo
    •  & Maria Grazia Spillantini

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Contributions

J.W.C. defined the direction of research. T.P.K., T.S.E., and R.J.E. designed all experiments. R.J.E. and V.B. designed the AAV vectors. V.B. performed the experiments in rat cortical preparations. T.P.K. performed the SCN slice experiments under the direction of M.H.H. L.C. performed experiments in live mice under the direction of M.G.S., E.C. performed experiments in live mice under the direction of M.T. T.S.E. provided ncAAs, analyzed lysates from live mice by fluorescent labeling and SDS-PAGE, performed the enrichments, and prepared samples for MS/MS. J.W.C., T.P.K., and R.J.E. wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jason W Chin.

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DOI

https://doi.org/10.1038/nbt.4056

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