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

Nature Biotechnology volume 35, pages 1196–1201 (2017)Cite this article

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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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Figure 1: Genetically targeted protein labeling.
Figure 2: Cell-type-specific proteomics in vivo: the hippocampal excitatory neuron proteome.
Figure 3: Cell-type-specific proteomics in vivo.
Figure 4: The CaMK2a excitatory hippocampal proteome in an enriched environment (EE).

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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

  1. Cyril Hanus

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

  2. Christoph T Schanzenbächer and Cyril Hanus: These authors contributed equally to this work.

Authors and 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,

    Anke Mueller & Daniela C Dieterich

  4. Leibniz Institute for Neurobiology, Magdeburg, Germany, Magdeburg, Germany

    Anke Mueller & Daniela C Dieterich

  5. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, USA

    David A Tirrell

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  1. Beatriz Alvarez-Castelao
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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.

Corresponding author

Correspondence to Erin M Schuman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 80295 kb)

Reporting Summary (PDF 159 kb)

Supplementary Table 1

CaMK2a (standard cage) excitatory hippocampal proteome. (XLSX 1098 kb)

Supplementary Table 2

Gene ontology: CaMK2a (standard cage) excitatoryhippocampal proteome. (XLSX 40 kb)

Supplementary Table 3

Cell-type specific protein and gene markers (XLSX 265 kb)

Supplementary Table 4

GAD2 proteome. (XLSX 764 kb)

Supplementary Table 5

Gene ontology: GAD2 Purkinje neuron proteome. (XLSX 360 kb)

Supplementary Table 6

Differentially expressed genes in CaMK2a (standard cage) vs GAD2 proteomes. (XLSX 289 kb)

Supplementary Table 7

CaMK2a enriched environment excitatory hippocampalproteome. (XLSX 1068 kb)

Supplementary Table 8

Differentially expressed genes in CaMK2a-standard cage vs CaMK2a- enriched environment proteomes. (XLSX 128 kb)

Supplementary Table 9

Gene ontology of differentially expressed genes inCaMK2a-standard cage vs CaMK2a-enriched environment proteomes. (XLSX 38 kb)

Supplementary Table 10

Primers used for mouse genotyping. (DOCX 59 kb)

Supplementary Table 11

Custom parameters used for MS analysis in MaxQuantand Perseus. (XLSX 20 kb)

Supplementary Table 12

Full hippocampus, cerebellum and glia proteomes. (XLSX 2312 kb)

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Alvarez-Castelao, B., Schanzenbächer, C., Hanus, C. et al. Cell-type-specific metabolic labeling of nascent proteomes in vivo. Nat Biotechnol 35, 1196–1201 (2017). https://doi.org/10.1038/nbt.4016

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