Letter

Cell-type-specific metabolic labeling of nascent proteomes in vivo

Received:
Accepted:
Published online:

Abstract

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

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Acknowledgements

We thank H. Geptin, D. Vogel, N. Fuerst, I. Wüllenweber and F. Rupprecht for excellent technical assistance. We thank E. Noll for the synthesis of ANL and P. Landgraf for the synthesis of the SH-alkyne. We thank S. Garg for her help with FUNCAT and M. Heumueller for his help with some of the experiments. We thank R. Pieaud and S. Junek for their assistance with imaging. We thank F. Kretschmer for his help with the open field analysis. We thank E. Northrup, S. Zeissler, S. Gil Mast and the animal facility of MPI for Brain Research for their excellent support. We thank J. Sanes and J. Chakkalakal for the early generation of a Thy-1 MetRS* mouse. Work in the laboratory of E.M.S. is supported by the Max Planck Society, the European Research Council, DFG CRC 902 and 1080, and the DFG Cluster of Excellence for Macromolecular Complexes. B.A. was supported by a Marie Curie Intra-European Fellowship for career development. C.H. was supported by a Marie Curie Career Integration Grant. D.C.D. is supported by DFG CRC 779 and 854. Research on proteomic labelling at Caltech is supported by the Programmable Molecular Technology Initiative of the Gordon and Betty Moore Foundation.

Author information

Author notes

    • Cyril Hanus

    Present address: Center for Psychiatry and Neurosciences, Inserm 894, Paris, France.

    • Christoph T Schanzenbächer
    •  & Cyril Hanus

    These authors contributed equally to this work.

Affiliations

  1. Max Planck Institute for Brain Research, Frankfurt, Germany.

    • Beatriz Alvarez-Castelao
    • , Christoph T Schanzenbächer
    • , Cyril Hanus
    • , Caspar Glock
    • , Susanne tom Dieck
    • , Aline R Dörrbaum
    • , Ina Bartnik
    • , Belquis Nassim-Assir
    • , Elena Ciirdaeva
    • , Julian D Langer
    •  & Erin M Schuman
  2. Max Planck Institute of Biophysics, Frankfurt, Germany.

    • Christoph T Schanzenbächer
    • , Aline R Dörrbaum
    •  & Julian D Langer
  3. Institute for Pharmacology and Toxicology, Otto von Guericke University, Magdeburg, Germany; Leibniz Institute for Neurobiology, Magdeburg, Germany; and Center for Behavioral Brain Sciences, Magdeburg, Germany.

    • Anke Mueller
    •  & Daniela C Dieterich
  4. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA.

    • David A Tirrell

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Contributions

B.A.-C., C.T.S. and C.H.: conception and design of experiments, acquisition, analysis and interpretation of data. C.G., S.t.D. and A.R.D.: conception and design of experiments, acquisition of data. I.B., B.N.-A. and E.C.: acquisition of data. A.M.: provided reagents. D.D.: provided reagents and advice on experiments. D.A.T.: conception and design of experiments. J.D.L.: acquisition of data, analysis and interpretation of data. E.M.S.: conception and design of experiments, analysis and interpretation of data, drafting, writing and revising the article. All authors contributed to the writing and revision of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Erin M Schuman.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

  2. 2.

    Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    CaMK2a (standard cage) excitatory hippocampal proteome.

  2. 2.

    Supplementary Table 2

    Gene ontology: CaMK2a (standard cage) excitatoryhippocampal proteome.

  3. 3.

    Supplementary Table 3

    Cell-type specific protein and gene markers

  4. 4.

    Supplementary Table 4

    GAD2 proteome.

  5. 5.

    Supplementary Table 5

    Gene ontology: GAD2 Purkinje neuron proteome.

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    Supplementary Table 6

    Differentially expressed genes in CaMK2a (standard cage) vs GAD2 proteomes.

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    Supplementary Table 7

    CaMK2a enriched environment excitatory hippocampalproteome.

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    Supplementary Table 8

    Differentially expressed genes in CaMK2a-standard cage vs CaMK2a- enriched environment proteomes.

  9. 9.

    Supplementary Table 9

    Gene ontology of differentially expressed genes inCaMK2a-standard cage vs CaMK2a-enriched environment proteomes.

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    Supplementary Table 11

    Custom parameters used for MS analysis in MaxQuantand Perseus.

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    Supplementary Table 12

    Full hippocampus, cerebellum and glia proteomes.

Word documents

  1. 1.

    Supplementary Table 10

    Primers used for mouse genotyping.