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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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References

  1. Sopko, R. & Perrimon, N. Receptor tyrosine kinases in Drosophila development. Cold Spring Harb. Perspect. Biol. 10.1101/cshperspect.a009050 (2013).

  2. Keene, A.C. & Sprecher, S.G. Seeing the light: photobehavior in fruit fly larvae. Trends Neurosci. 35, 104–110 (2012).

    Article  CAS  PubMed  Google Scholar 

  3. Tepass, U. The apical polarity protein network in Drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival. Annu. Rev. Cell Dev. Biol. 28, 655–685 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Dubnau, J. Ode to the mushroom bodies. Science 335, 664–665 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Ngo, J.T. & Tirrell, D.A. Noncanonical amino acids in the interrogation of cellular protein synthesis. Acc. Chem. Res. 44, 677–685 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Howden, A.J. et al. QuaNCAT: quantitating proteome dynamics in primary cells. Nat. Methods 10, 343–346 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ngo, J.T. et al. Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Truong, F., Yoo, T.H., Lampo, T.J. & Tirrell, D.A. Two-strain, cell-selective protein labeling in mixed bacterial cultures. J. Am. Chem. Soc. 134, 8551–8556 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ngo, J.T., Schuman, E.M. & Tirrell, D.A. Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells. Proc. Natl. Acad. Sci. USA 110, 4992–4997 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hinz, F.I., Dieterich, D.C., Tirrell, D.A. & Schuman, E.M. Non-canonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish. ACS Chem. Neurosci. 3, 40–49 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Link, A.J. et al. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc. Natl. Acad. Sci. USA 103, 10180–10185 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Chin, J.W. Reprogramming the genetic code. Science 336, 428–429 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Davis, L. & Chin, J.W. Designer proteins: applications of genetic code expansion in cell biology. Nat. Rev. Mol. Cell Biol. 13, 168–182 (2012).

    Article  CAS  PubMed  Google Scholar 

  14. Neumann, H., Peak-Chew, S.Y. & Chin, J.W. Genetically encoding N(epsilon)-acetyllysine in recombinant proteins. Nat. Chem. Biol. 4, 232–234 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Hancock, S.M., Uprety, R., Deiters, A. & Chin, J.W. Expanding the genetic code of yeast for incorporation of diverse unnatural amino acids via a pyrrolysyl-tRNA synthetase/tRNA pair. J. Am. Chem. Soc. 132, 14819–14824 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Mukai, T. et al. Adding l–lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases. Biochem. Biophys. Res. Commun. 371, 818–822 (2008).

    Article  CAS  PubMed  Google Scholar 

  17. Chen, P.R. et al. A facile system for encoding unnatural amino acids in mammalian cells. Angew. Chem. Int. Ed. 48, 4052–4055 (2009).

    Article  CAS  Google Scholar 

  18. Gautier, A. et al. Genetically encoded photocontrol of protein localization in mammalian cells. J. Am. Chem. Soc. 132, 4086–4088 (2010).

    Article  CAS  PubMed  Google Scholar 

  19. Greiss, S. & Chin, J.W. Expanding the genetic code of an animal. J. Am. Chem. Soc. 133, 14196–14199 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Bianco, A., Townsley, F.M., Greiss, S., Lang, K. & Chin, J.W. Expanding the genetic code of Drosophila melanogaster. Nat. Chem. Biol. 8, 748–750 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Blackman, M.L., Royzen, M. & Fox, J.M. Tetrazine ligation: fast bioconjugation based on inverse-electron-demand Diels−Alder reactivity. J. Am. Chem. Soc. 130, 13518–13519 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yang, J., Šečćkutė, J., Cole, C.M. & Devaraj, N.K. Live-cell imaging of cyclopropene tags with fluorogenic tetrazine cycloadditions. Angew. Chem. Int. Engl.Ed. 51, 7476–7479 (2012).

    Article  CAS  Google Scholar 

  23. Cole, C.M., Yang, J., Šečćkutė, J. & Devaraj, N.K. Fluorescent live-cell imaging of metabolically incorporated unnatural cyclopropene-mannosamine derivatives. ChemBioChem 14, 205–208 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Patterson, D.M., Nazarova, L.A., Xie, B., Kamber, D.N. & Prescher, J.A. Functionalized cyclopropenes as bioorthogonal chemical reporters. J. Am. Chem. Soc. 134, 18638–18643 (2012).

    Article  CAS  PubMed  Google Scholar 

  25. Devaraj, N.K. & Weissleder, R. Biomedical applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816–827 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Lang, K. et al. Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction. Nat. Chem. 4, 298–304 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lang, K. et al. Genetic encoding of bicyclononynes and trans-cyclooctenes for site-specific protein labeling in vitro and in live mammalian cells via rapid fluorogenic Diels–Alder reactions. J. Am. Chem. Soc. 134, 10317–10320 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Borrmann, A. et al. Genetic encoding of a bicyclo[6.1.0]nonyne-charged amino acid enables fast cellular protein imaging by metal-free ligation. ChemBioChem 13, 2094–2099 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Plass, T. et al. Amino acids for Diels–Alder reactions in living cells. Angew. Chem. Int. Ed. Engl. 51, 4166–4170 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Seitchik, J.L. et al. Genetically encoded tetrazine amino acid directs rapid site-specific in vivo bioorthogonal ligation with trans-cyclooctenes. J. Am. Chem. Soc. 134, 2898–2901 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Nguyen, D.P. et al. Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA synthetase/tRNACUA pair and click chemistry. J. Am. Chem. Soc. 131, 8720–8721 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Yu, Z., Pan, Y., Wang, Z., Wang, J. & Lin, Q. Genetically encoded cyclopropene directs rapid, photoclick-chemistry-mediated protein labeling in mammalian cells. Angew. Chem. Int. Ed. Engl. 51, 10600–10604 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Virdee, S., Ye, Y., Nguyen, D.P., Komander, D. & Chin, J.W. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat. Chem. Biol. 6, 750–757 (2010).

    Article  CAS  PubMed  Google Scholar 

  34. Ambrogelly, A. et al. Pyrrolysine is not hardwired for cotranslational insertion at UAG codons. Proc. Natl. Acad. Sci. USA 104, 3141–3146 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63 (1983).

    Article  CAS  PubMed  Google Scholar 

  36. Brand, A.H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  PubMed  Google Scholar 

  37. Hadjieconomou, D. et al. Flybow: genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Nat. Methods 8, 260–266 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Wu, J.S. & Luo, L. A protocol for mosaic analysis with a repressible cell marker (MARCM) in Drosophila. Nat. Protoc. 1, 2583–2589 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Wang, C., Dickinson, L.K. & Lehmann, R. Genetics of nanos localization in Drosophila. Dev. Dyn. 199, 103–115 (1994).

    Article  CAS  PubMed  Google Scholar 

  40. Fox, D.T. & Duronio, R.J. Endoreplication and polyploidy: insights into development and disease. Development 140, 3–12 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Von Stetina, J.R., Lafever, K.S., Rubin, M. & Drummond-Barbosa, D. A genetic screen for dominant enhancers of the cell-cycle regulator alpha-endosulfine identifies matrimony as a strong functional interactor in Drosophila. G3 (Bethesda) 1, 607–613 (2011).

    Article  CAS  Google Scholar 

  42. Feret, R. & Lilley, K. S. Protein profiling using two-dimensional difference gel electrophoresis (2-D Dige). Curr. Protoc. Protein Sci. 75, 22.2 (2014).

    Google Scholar 

  43. Bownes, M. Expression of the genes coding for vitellogenin (yolk protein). Annu. Rev. Entomol. 31, 507–531 (1986).

    Article  CAS  Google Scholar 

  44. Graveley, B.R. et al. The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011).

    Article  CAS  PubMed  Google Scholar 

  45. Leon, A. & McKearin, D. Identification of TER94, an AAA ATPase protein, as a Bam-dependent component of the Drosophila fusome. Mol. Biol. Cell 10, 3825–3834 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Liang, L., Diehl-Jones, W. & Lasko, P. Localization of vasa protein to the Drosophila pole plasm is independent of its RNA-binding and helicase activities. Development 120, 1201–1211 (1994).

    CAS  PubMed  Google Scholar 

  47. Suchanek, M., Radzikowska, A. & Thiele, C. Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat. Methods 2, 261–268 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Karp, N.A., Kreil, D.P. & Lilley, K.S. Determining a significant change in protein expression with DeCyder during a pair-wise comparison using two-dimensional difference gel electrophoresis. Proteomics 4, 1421–1432 (2004).

    Article  CAS  PubMed  Google Scholar 

  49. Harper, S., Mozdzanowski, J. & Speicher, D . Two-dimensional gel electrophoresis. Curr. Protoc. Protein Sci. 11, 10.4 (2001).

    Google Scholar 

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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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