Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published December 2017 | Supplemental Material
Journal Article Open

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

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.

Additional Information

© 2017 Macmillan Publishers Limited, part of Springer Nature. Received: 12 December 2016; Accepted: 19 October 2017; Published online: 06 November 2017. 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 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. The authors declare no competing financial interests.

Attached Files

Supplemental Material - nbt.4016-S1.pdf

Supplemental Material - nbt.4016-S10.xlsx

Supplemental Material - nbt.4016-S11.xlsx

Supplemental Material - nbt.4016-S12.docx

Supplemental Material - nbt.4016-S13.xlsx

Supplemental Material - nbt.4016-S14.xlsx

Supplemental Material - nbt.4016-S2.pdf

Supplemental Material - nbt.4016-S3.xlsx

Supplemental Material - nbt.4016-S4.xlsx

Supplemental Material - nbt.4016-S5.xlsx

Supplemental Material - nbt.4016-S6.xlsx

Supplemental Material - nbt.4016-S7.xlsx

Supplemental Material - nbt.4016-S8.xlsx

Supplemental Material - nbt.4016-S9.xlsx

Files

nbt.4016-S2.pdf
Files (30.1 MB)
Name Size Download all
md5:e0a818b350044b11f122665f5a048d74
2.4 MB Download
md5:218d30df6afb74d8a2da84d3b18a3ed5
60.9 kB Download
md5:588fd620da8cf6e60089eb884f453594
21.3 kB Download
md5:109ffdde5a7adf161e882458a8750a2d
162.9 kB Preview Download
md5:cfad73525bfce8dad5a1b72560126350
131.3 kB Download
md5:0171e372ae38b905cda4c15235f3a746
1.1 MB Download
md5:a383856400513ea4b9d45c89a727729d
272.3 kB Download
md5:685d5e2558bacb7d27a10119442cee92
782.6 kB Download
md5:33657c12ac65e70a7d6cef5e515ae61f
23.4 MB Preview Download
md5:d49eb6df63772355eb1a56dc0cd8ddaf
41.9 kB Download
md5:9a614867364abdc8e1b627c5b35c5900
369.6 kB Download
md5:4ed9017362f4ba463ab5488dc4dcf24c
296.0 kB Download
md5:a333fe4839c0539bd4b8705309039ad0
39.3 kB Download
md5:ae30cf929ff4cfe23ce0918ce631bc6b
1.1 MB Download

Additional details

Created:
August 19, 2023
Modified:
October 17, 2023