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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The datasets presented in this Protocol were originally generated in ref. 12. All data are available from the corresponding author on reasonable request.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
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.