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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Chimeric design of pyrrolysyl-tRNA synthetase/tRNA pairs and canonical synthetase/tRNA pairs for genetic code expansion
Nature Communications Open Access 22 June 2020
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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.
The authors declare no competing financial interests.
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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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