Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo

Abstract

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ANL incorporation into the mutant MetRS and mouse design.
Fig. 2: Workflow for cell-type-specific protein labeling and purification.
Fig. 3: Dosage of ANL in drinking water.
Fig. 4: Alkyne dosage testing.
Fig. 5: Cell-type-specific protein purification.
Fig. 6: Analysis of eluted proteins.
Fig. 7: Methionine content of the eluted peptides.

Similar content being viewed by others

Data availability

The datasets presented in this Protocol were originally generated in ref. 12. All data are available from the corresponding author on reasonable request.

References

  1. Feist, P. & Hummon, A. B. Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int. J. Mol. Sci. 16, 3537–3563 (2015).

    Article  CAS  Google Scholar 

  2. McKay, C. S. & Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075–1101 (2014).

    Article  CAS  Google Scholar 

  3. Laughlin, S. T., Baskin, J. M., Amacher, S. L. & Bertozzi, C. R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    Article  CAS  Google Scholar 

  4. Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A. & Schuman, E. M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

    Article  CAS  Google Scholar 

  5. Elsasser, S. J., Ernst, R. J., Walker, O. S. & Chin, J. W. Genetic code expansion in stable cell lines enables encoded chromatin modification. Nat. Methods 13, 158–164 (2016).

    Article  CAS  Google Scholar 

  6. Ngo, J. T. et al. Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 (2009).

    Article  CAS  Google Scholar 

  7. Mahdavi, A. et al. Engineered aminoacyl-tRNA synthetase for cell-selective analysis of mammalian protein synthesis. J. Am. Chem. Soc. 138, 4278–4281 (2016).

    Article  CAS  Google Scholar 

  8. Yuet, K. P. et al. Cell-specific proteomic analysis in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 112, 2705–2710 (2015).

    Article  CAS  Google Scholar 

  9. Link, A. J. et al. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc. Natl. Acad. Sci. USA 103, 10180–10185 (2006).

    Article  CAS  Google Scholar 

  10. de Felipe, P. et al. E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol. 24, 68–75 (2006).

    Article  Google Scholar 

  11. Griffin, R. J. The medicinal chemistry of the azido group. Prog. Med. Chem. 31, 121–232 (1994).

    Article  CAS  Google Scholar 

  12. Alvarez-Castelao, B. et al. Cell-type-specific metabolic labeling of nascent proteomes in vivo. Nat. Biotechnol. 35, 1196–1201 (2017).

    Article  CAS  Google Scholar 

  13. Bennett, E. L., Diamond, M. C., Krech, D. & Rosenzweig, M. R. Chemical and anatomical plasticity brain. Science 146, 610–619 (1964).

    Article  CAS  Google Scholar 

  14. Dieterich, D. C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat. Neurosci. 13, 897–905 (2010).

    Article  CAS  Google Scholar 

  15. tom Dieck, S. et al. Direct visualization of newly synthesized target proteins in situ. Nat. Methods 12, 411–414 (2015).

    Article  CAS  Google Scholar 

  16. Liu, Y. et al. Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis. Nat. Commun. 8, 643 (2017).

    Article  Google Scholar 

  17. Liu, Y. et al. Addendum: application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis. Nat. Commun. 9, 1052 (2018).

    Article  Google Scholar 

  18. Zanivan, S., Krueger, M. & Mann, M. In vivo quantitative proteomics: the SILAC mouse. Methods Mol. Biol. 757, 435–450 (2012).

    Article  Google Scholar 

  19. Fornasiero, E. F. et al. Precisely measured protein lifetimes in the mouse brain reveal differences across tissues and subcellular fractions. Nat. Commun. 9, 4230 (2018).

    Article  Google Scholar 

  20. Gauthier, N. P. et al. Cell-selective labeling using amino acid precursors for proteomic studies of multicellular environments. Nat. Methods 10, 768–773 (2013).

    Article  CAS  Google Scholar 

  21. Jansens, A. & Braakman, I. Pulse-chase labeling techniques for the analysis of protein maturation and degradation. Methods Mol. Biol. 232, 133–145 (2003).

    CAS  PubMed  Google Scholar 

  22. Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6, 275–277 (2009).

    Article  CAS  Google Scholar 

  23. Goodman, C. A. & Hornberger, T. A. Measuring protein synthesis with SUnSET: a valid alternative to traditional techniques? Exerc. Sport Sci. Rev. 41, 107–115 (2013).

    Article  Google Scholar 

  24. Starck, S. R., Green, H. M., Alberola-Ila, J. & Roberts, R. W. A general approach to detect protein expression in vivo using fluorescent puromycin conjugates. Chem. Biol. 11, 999–1008 (2004).

    Article  CAS  Google Scholar 

  25. Marciano, R., Leprivier, G. & Rotblat, B. Puromycin labeling does not allow protein synthesis to be measured in energy-starved cells. Cell Death Dis. 9, 39 (2018).

    Article  Google Scholar 

  26. Du, S. et al. Cell type-selective imaging and profiling of newly synthesized proteomes by using puromycin analogues. Chem. Commun. 53, 8443–8446 (2017).

    Article  CAS  Google Scholar 

  27. Barrett, R. M., Liu, H. W., Jin, H., Goodman, R. H. & Cohen, M. S. Cell-specific profiling of nascent proteomes using orthogonal enzyme-mediated puromycin incorporation. ACS Chem. Biol. 11, 1532–1536 (2016).

    Article  CAS  Google Scholar 

  28. McShane, E. et al. Kinetic analysis of protein stability reveals age-dependent degradation. Cell 167, 803–815.e21 (2016).

    Article  CAS  Google Scholar 

  29. McClatchy, D. B. et al. Pulsed azidohomoalanine labeling in mammals (PALM) detects changes in liver-specific LKB1 knockout mice. J. Proteome Res. 14, 4815–4822 (2015).

    Article  CAS  Google Scholar 

  30. Alvarez-Castelao, B. & Schuman, E. M. The regulation of synaptic protein turnover. J. Biol. Chem. 290, 28623–28630 (2015).

    Article  CAS  Google Scholar 

  31. Woodruff-Pak, D. S. Stereological estimation of Purkinje neuron number in C57BL/6 mice and its relation to associative learning. Neuroscience 141, 233–243 (2006).

    Article  CAS  Google Scholar 

  32. Link, A. J., Vink, M. K. & Tirrell, D. A. Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer. Nat. Protoc. 2, 1879–1883 (2007).

    Article  CAS  Google Scholar 

  33. Szychowski, J. et al. Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition. J. Am. Chem. Soc. 132, 18351–18360 (2010).

    Article  CAS  Google Scholar 

  34. Gillet, L. C., Leitner, A. & Aebersold, R. Mass spectrometry applied to bottom-up proteomics: entering the high-throughput era for hypothesis testing. Annu. Rev. Anal. Chem. 9, 449–472 (2016).

    Article  Google Scholar 

  35. Landgraf, P., Antileo, E. R., Schuman, E. M. & Dieterich, D. C. BONCAT: metabolic labeling, click chemistry, and affinity purification of newly synthesized proteomes. Methods Mol. Biol. 1266, 199–215 (2015).

    Article  CAS  Google Scholar 

  36. Schanzenbächer, C. T., Langer, J. D. & Schuman, E. M. Time- and polarity-dependent proteomic changes associated with homeostatic scaling at central synapses. Elife 7, e33322 (2018).

    Article  Google Scholar 

  37. Schanzenbächer, C. T., Sambandan, S., Langer, J. D. & Schuman, E. M. Nascent proteome remodeling following homeostatic scaling at hippocampal synapses. Neuron 92, 358–371 (2016).

    Article  Google Scholar 

  38. Wessel, D. & Flugge, U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143 (1984).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank C. Hanus, C. Glock, S. tom Dieck, A.R. Dörrbaum, I. Bartnik, B. Nassim-Assir, E. Ciirdaeva, A. Mueller, D.C. Dieterich and D.A. Tirrell for their contributions to the Nature Biotechnology paper12. We thank H. Geptin, D. Vogel., N. Fürst, I. Wüllenweber and F. Rupprecht for their excellent technical assistance. We thank E. Noll for the synthesis of ANL and P. Landgraf for the synthesis of the DST alkyne. We thank E. Northrup, S. Zeissler, S. Gil Mast and the animal facility of the MPI for Brain Research for their excellent support. Work in the laboratory of E.M.S. was supported by the Max Planck Society, the European Research Council, grants DFG CRC 902 and 1080, and the DFG Cluster of Excellence for Macromolecular Complexes; this project received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 743216). B.A.-C. was supported by a Marie Curie IEF grant.

Author information

Authors and Affiliations

Authors

Contributions

B.A.-C. and C.T.S. designed the experiments, and acquired, analyzed and interpreted the data. J.D.L. and E.M.S. designed the experiments, and analyzed and interpreted the data. E.M.S. and B.A.-C. wrote and revised the manuscript. All authors contributed to the writing and revision of the article.

Corresponding authors

Correspondence to Beatriz Alvarez-Castelao or Erin M. Schuman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Related links

Key references using this protocol

Alvarez-Castelao, B. et al. Nat. Biotechnol. 35, 1196–1201 (2017): https://www.nature.com/articles/nbt.4016

Yuet, K. P. et al. Proc. Natl. Acad. Sci. USA 112, 2705–2710 (2015): http://www.pnas.org/content/112/9/2705

Ngo, J. T. et al. Nat. Chem. Biol. 5, 715–717 (2009): https://www.nature.com/articles/nchembio.200

Integrated supplementary information

Supplementary Figure 1 Sypro Ruby staining of eluted proteins.

Gel stained with Sypro Ruby showing 3 biological replicates of cell-type specific eluted proteins derived from the negative control (wt mice) and Camk2-Cre::R26-MetRS* mice, labeled during 21 days with 1% of ANL administered in the drinking water. The hippocampus was dissected and used for the experiment. Adapted with permission from Alvarez-Castelao et al.12, Springer Nature.

Supplementary Figure 2 Example of a failed biological replicate.

a, Plot showing similar abundance in Camk2-Cre::R26-MetRS* compared with WT mouse samples of proteins found in both groups (peptide intensities). b, Union of proteins unique to or markedly enriched (>3-fold WT) in Camk2-Cre::R26-MetRS* mice, showing a very low number of proteins (526).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2 and Supplementary Methods

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alvarez-Castelao, B., Schanzenbächer, C.T., Langer, J.D. et al. Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo. Nat Protoc 14, 556–575 (2019). https://doi.org/10.1038/s41596-018-0106-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-018-0106-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research