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Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal

Nature Biotechnology volume 32, pages 465–472 (2014)Cite this article

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Abstract

Identifying the proteins synthesized at specific times in cells of interest in an animal will facilitate the study of cellular functions and dynamic processes. Here we introduce stochastic orthogonal recoding of translation with chemoselective modification (SORT-M) to address this challenge. SORT-M involves modifying cells to express an orthogonal aminoacyl-tRNA synthetase/tRNA pair to enable the incorporation of chemically modifiable analogs of amino acids at diverse sense codons in cells in rich media. We apply SORT-M to Drosophila melanogaster fed standard food to label and image proteins in specific tissues at precise developmental stages with diverse chemistries, including cyclopropene-tetrazine inverse electron demand Diels-Alder cycloaddition reactions. We also use SORT-M to identify proteins synthesized in germ cells of the fly ovary without dissection. SORT-M will facilitate the definition of proteins synthesized in specific sets of cells to study development, and learning and memory in flies, and may be extended to other animals.

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Figure 1: SORT-M enables proteome tagging and labeling at diverse codons, with diverse chemistries, and in genetically targeted cells and tissues.
Figure 2: Site-specific incorporation of 3 into proteins at diverse codons and specific proteome labeling using SORT-M in human cells.
Figure 3: Site-specific incorporation of amino acid 3 into protein produced in Drosophila melanogaster.
Figure 4: SORT-M enables selective imaging of proteins synthesized within the germ cells of the fly ovary from stage 5 onwards.
Figure 5: SORT-M facilitates tissue-specific labeling of the fly proteome.
Figure 6: Tissue- and developmental stage–specific proteome labeling, protein identification and validation.

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Acknowledgements

We are exceptionally grateful to M. Skehl and S. Maslen of the LMB Mass Spectrometry service for mass spectrometry, R. Feret of the Cambridge Centre for Proteomics for assistance with DIGE, and S. Bullock (LMB) for advice and comments. We are grateful to the Herchel Smith Fund (S.J.E., administered through Cambridge University Department of Chemistry) the Medical Research Council (U105181009, UD99999908) and the European Research Council for funding.

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Author notes

  1. Thomas S Elliott, Fiona M Townsley and Ambra Bianco: These authors contributed equally to this work.

Authors and Affiliations

  1. Medical Research Council Laboratory of Molecular Biology, Cambridge, England, UK

    Thomas S Elliott, Fiona M Townsley, Ambra Bianco, Russell J Ernst, Amit Sachdeva, Simon J Elsässer, Lloyd Davis, Kathrin Lang, Rudolf Pisa, Sebastian Greiss & Jason W Chin

  2. Department of Chemistry, University of Cambridge, Cambridge, England, UK

    Rudolf Pisa & Jason W Chin

  3. Department of Biochemistry, University of Cambridge, Cambridge, England, UK

    Kathryn S Lilley

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Contributions

T.S.E. developed the chemistry for SORT-M in E. coli with contributions from K.L., A.S. and R.J.E. F.M.T., A.B. and T.S.E. developed SORT-M fly approach. R.J.E. developed SORT-M in mammalian cells with contributions from S.J.E. and L.D. R.P. did initial experiments using 2, with initial input from S.G. K.S.L. provided 2D-DIGE expertise. J.W.C. supervised the project. J.W.C., T.S.E., F.M.T. and A.B. wrote the paper with input from other authors.

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Correspondence to Jason W Chin.

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Elliott, T., Townsley, F., Bianco, A. et al. Proteome labeling and protein identification in specific tissues and at specific developmental stages in an animal. Nat Biotechnol 32, 465–472 (2014). https://doi.org/10.1038/nbt.2860

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