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


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).

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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.

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.


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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.

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Authors and Affiliations



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.

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The authors declare no competing interests.

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Key references using this protocol

Alvarez-Castelao, B. et al. Nat. Biotechnol. 35, 1196–1201 (2017):

Yuet, K. P. et al. Proc. Natl. Acad. Sci. USA 112, 2705–2710 (2015):

Ngo, J. T. et al. Nat. Chem. Biol. 5, 715–717 (2009):

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).

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Supplementary Figures 1 and 2 and Supplementary Methods

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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).

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