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Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans

A Corrigendum to this article was published on 20 November 2014

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Abstract

In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo–synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo–synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1–2 d for bio-orthogonal labeling; and, with regard to analysis, 3–4 d for western blotting, 5–6 d for immunofluorescence or 3 weeks for mass spectrometry.

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Figure 1: Bio-orthogonal labeling workflow.
Figure 2: Protocol workflow.
Figure 3: Images of an adult C. elegans and L1 and L4 larvae by bright-field microscopy of mounted specimen.
Figure 4: Testing for AHA toxicity—the thrashing assay.
Figure 5: Testing for AHA toxicity—the unfolded protein response (UPR) assay.
Figure 6: Copper-catalyzed azide-alkyne cyclo-addition.
Figure 7: Detection of AHA incorporation by western blotting.
Figure 8: Detection of azidohomoalanine (AHA) incorporation by fluorescence microscopy.
Figure 9: iTRAQ quantitative proteomic data from an MS/MS spectrum performed to detect peptides that have incorporated AHA.

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  • 12 September 2014

     In the version of this article initially published, one of the two affiliations of one of the authors (Michael Kassiou) was incorrect. The mistaken affiliation read: “6Faculty of Health Sciences, Macquarie University, Sydney, New South Wales, Australia.” The correct affiliation is: “6Faculty of Health Sciences, University of Sydney, Sydney, New South Wales, Australia.” The error has been corrected in the HTML and PDF versions of the article.

References

  1. Taylor, R.C. & Dillin, A. Aging as an event of proteostasis collapse. Cold Spring Harb. Perspect. Biol. 3 (2011).

  2. Kaeberlein, M. & Kennedy, B.K. Hot topics in aging research: protein translation and TOR signaling, 2010. Aging Cell 10, 185–190 (2011).

    Article  CAS  Google Scholar 

  3. Giboureau, N., Som, I.M., Boucher-Arnold, A., Guilloteau, D. & Kassiou, M. PET radioligands for the vesicular acetylcholine transporter (VAChT). Curr. Top Med. Chem. 10, 1569–1583 (2010).

    Article  CAS  Google Scholar 

  4. De Strooper, B. Proteases and proteolysis in Alzheimer disease: a multifactorial view on the disease process. Physiol. Rev. 90, 465–494 (2010).

    Article  CAS  Google Scholar 

  5. Chen, C.C. et al. Visualizing long-term memory formation in two neurons of the Drosophila brain. Science 335, 678–685 (2012).

    Article  CAS  Google Scholar 

  6. Götz, J., Chen, F., van Dorpe, J. & Nitsch, R.M. Formation of neurofibrillary tangles in P301L tau transgenic mice induced by Aβ42 fibrils. Science 293, 1491–1495 (2001).

    Article  Google Scholar 

  7. Ittner, L.M. et al. Dendritic function of tau mediates amyloid-β toxicity in Alzheimer's disease mouse models. Cell 142, 387–397 (2010).

    Article  CAS  Google Scholar 

  8. Cajigas, I.J., Will, T. & Schuman, E.M. Protein homeostasis and synaptic plasticity. EMBO J. 29, 2746–2752 (2010).

    Article  CAS  Google Scholar 

  9. Antonov, I., Kandel, E.R. & Hawkins, R.D. Presynaptic and postsynaptic mechanisms of synaptic plasticity and metaplasticity during intermediate-term memory formation in Aplysia. J. Neurosci. 30, 5781–5791 (2010).

    Article  CAS  Google Scholar 

  10. Holt, C.E. & Schuman, E.M. The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80, 648–657 (2013).

    Article  CAS  Google Scholar 

  11. David, D.C. et al. Proteomic and functional analysis reveal a mitochondrial dysfunction in P301L tau transgenic mice. J. Biol. Chem. 280, 23802–23814 (2005).

    Article  CAS  Google Scholar 

  12. David, D.C. et al. β-Amyloid treatment of two complementary P301L tau-expressing Alzheimer's disease models reveals similar deregulated cellular processes. Proteomics 6, 6566–6577 (2006).

    Article  CAS  Google Scholar 

  13. Lim, Y.-A. et al. Aβ and human amylin share a common toxicity pathway via mitochondrial dysfunction. Proteomics 10, 1621–1633 (2010).

    Article  CAS  Google Scholar 

  14. Rhein, V. et al. Amyloid-β and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice. Proc. Natl. Acad. Sci. USA 106, 20057–20062 (2009).

    Article  CAS  Google Scholar 

  15. Chen, F. et al. Role for glyoxalase I in Alzheimer's disease. Proc. Natl. Acad. Sci. USA 101, 7687–7692 (2004).

    Article  CAS  Google Scholar 

  16. Hoerndli, F.J., Pelech, S., Papassotiropoulos, A. & Götz, J. Aβ treatment and P301L tau expression in an Alzheimer's disease tissue culture model act synergistically to promote aberrant cell cycle re-entry. Eur. J. Neurosci. 26, 60–72 (2007).

    Article  Google Scholar 

  17. Schonrock, N. et al. Neuronal microRNA deregulation in response to Alzheimer's disease amyloid-β. PLoS ONE 5, e11070 (2010).

    Article  Google Scholar 

  18. Götz, J. & Ittner, L.M. Animal models of Alzheimer's disease and frontotemporal dementia. Nat. Rev. Neurosci. 9, 532–544 (2008).

    Article  Google Scholar 

  19. Chew, Y.L., Fan, X., Götz, J. & Nicholas, H.R. Protein with tau-like repeats regulates neuronal integrity and lifespan in C. elegans. J. Cell Sci. 126, 2079–2091 (2013).

    Article  CAS  Google Scholar 

  20. Pienaar, I.S., Götz, J. & Feany, M.B. Parkinson's disease: insights from non-traditional model organisms. Prog. Neurobiol. 92, 558–571 (2010).

    Article  CAS  Google Scholar 

  21. Duboff, B., Götz, J. & Feany, M.B. Tau promotes neurodegeneration via DRP1 mislocalization. Neuron 75, 618–632 (2012).

    Article  CAS  Google Scholar 

  22. Liang, V. et al. Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome. Cell Mol. Life Sci. 10.1007/s00018-014-1558-7 (2014).

  23. Kaletta, T. & Hengartner, M.O. Finding function in novel targets: C. elegans as a model organism. Nat. Rev. Drug Discov. 5, 387–398 (2006).

    Article  CAS  Google Scholar 

  24. Gautier, A., Nakata, E., Lukinavicius, G., Tan, K.T. & Johnsson, K. Selective cross-linking of interacting proteins using self-labeling tags. J. Am. Chem. Soc. 131, 17954–17962 (2009).

    Article  CAS  Google Scholar 

  25. Gautier, A. et al. An engineered protein tag for multiprotein labeling in living cells. Chem. Biol. 15, 128–136 (2008).

    Article  CAS  Google Scholar 

  26. Machleidt, T., Robers, M. & Hanson, G.T. Protein labeling with FlAsH and ReAsH. Methods Mol. Biol. 356, 209–220 (2007).

    CAS  PubMed  Google Scholar 

  27. Hughes, A.J. & Herr, A.E. Microfluidic western blotting. Proc. Natl. Acad. Sci. USA 109, 21450–21455 (2012).

    Article  CAS  Google Scholar 

  28. David, D., Hoerndli, F. & Götz, J. Functional genomics meets neurodegenerative disorders. Part I: transcriptomic and proteomic technology. Prog. Neurobiol. 76, 153–168 (2005).

    Article  CAS  Google Scholar 

  29. Gygi, S.P. et al. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17, 994–999 (1999).

    Article  CAS  Google Scholar 

  30. Husi, H. et al. Selective chemical intervention in the proteome of Caenorhabditis elegans. J. Proteome Res. 9, 6060–6070 (2010).

    Article  CAS  Google Scholar 

  31. Fredens, J. et al. Quantitative proteomics by amino acid labeling in C. elegans. Nat. Methods 8, 845–847 (2011).

    Article  CAS  Google Scholar 

  32. Larance, M. et al. Stable-isotope labeling with amino acids in nematodes. Nat. Methods 8, 849–851 (2011).

    Article  CAS  Google Scholar 

  33. Boisvert, F.M. et al. A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol. Cell. Proteomics 11, M111.011429 (2012).

    Article  Google Scholar 

  34. Seyfried, N.T. et al. Multiplex SILAC analysis of a cellular TDP-43 proteinopathy model reveals protein inclusions associated with SUMOylation and diverse polyubiquitin chains. Mol. Cell. Proteomics 9, 705–718 (2010).

    Article  CAS  Google Scholar 

  35. Huh, K.H. & Wenthold, R.J. Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells. J. Biol. Chem. 274, 151–157 (1999).

    Article  CAS  Google Scholar 

  36. Shi, H.J., Stubbs, R. & Hood, K. Characterization of de novo synthesized proteins released from human colorectal tumour explants. Electrophoresis 30, 2442–2453 (2009).

    Article  CAS  Google Scholar 

  37. Ong, S.E. & Mann, M. A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660 (2006).

    Article  CAS  Google Scholar 

  38. Prescher, J.A. & Bertozzi, C.R. Chemistry in living systems. Nat. Chem. Biol. 1, 13–21 (2005).

    Article  CAS  Google Scholar 

  39. Beatty, K.E. & Tirrell, D.A. Two-color labeling of temporally defined protein populations in mammalian cells. Bioorg. Med. Chem. Lett. 18, 5995–5999 (2008).

    Article  CAS  Google Scholar 

  40. Kolb, H.C., Finn, M.G. & Sharpless, K.B. Click chemistry: diverse chemical function from a few good reactions. Angew Chem. Int. Ed. Engl. 40, 2004–2021 (2001).

    Article  CAS  Google Scholar 

  41. Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A. & Schuman, E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

    Article  CAS  Google Scholar 

  42. Kiick, K.L., Saxon, E., Tirrell, D.A. & Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).

    Article  CAS  Google Scholar 

  43. Havrylenko, S., Legouis, R., Negrutskii, B. & Mirande, M. Methionyl-tRNA synthetase from Caenorhabditis elegans: a specific multidomain organization for convergent functional evolution. Protein Sci. 19, 2475–2484 (2010).

    Article  CAS  Google Scholar 

  44. Laughlin, S.T., Baskin, J.M., Amacher, S.L. & Bertozzi, C.R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    Article  CAS  Google Scholar 

  45. Kho, Y. et al. A tagging-via-substrate technology for detection and proteomics of farnesylated proteins. Proc. Natl. Acad. Sci. USA 101, 12479–12484 (2004).

    Article  CAS  Google Scholar 

  46. Bruckman, M.A. et al. Surface modification of tobacco mosaic virus with 'click' chemistry. Chembiochem 9, 519–523 (2008).

    Article  CAS  Google Scholar 

  47. Weisbrod, S.H. & Marx, A. Novel strategies for the site-specific covalent labelling of nucleic acids. Chem. Commun. (Camb.) 2008, 5675–5685 (2008).

    Article  Google Scholar 

  48. 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  Google Scholar 

  49. Laughlin, S.T. & Bertozzi, C.R. Imaging the glycome. Proc. Natl. Acad. Sci. USA 106, 12–17 (2009).

    Article  CAS  Google Scholar 

  50. Laughlin, S.T. & Bertozzi, C.R. In vivo imaging of Caenorhabditis elegans glycans. ACS Chem. Biol. 4, 1068–1072 (2009).

    Article  CAS  Google Scholar 

  51. Dieterich, D.C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).

    Article  Google Scholar 

  52. Dieterich, D.C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat. Neurosci. 13, 897–905 (2010).

    Article  CAS  Google Scholar 

  53. Putz, S.M., Boehm, A.M., Stiewe, T. & Sickmann, A. iTRAQ analysis of a cell culture model for malignant transformation, including comparison with 2D-PAGE and SILAC. J. Proteome Res. 11, 2140–2153 (2012).

    Article  Google Scholar 

  54. Yoon, B.C. et al. Local translation of extranuclear lamin B promotes axon maintenance. Cell 148, 752–764 (2012).

    Article  CAS  Google Scholar 

  55. Nicholas, H.R. & Hodgkin, J. The ERK MAP kinase cascade mediates tail swelling and a protective response to rectal infection in C. elegans. Curr. Biol. 14, 1256–1261 (2004).

    Article  CAS  Google Scholar 

  56. Nicholas, H.R. & Hodgkin, J. Responses to infection and possible recognition strategies in the innate immune system of Caenorhabditis elegans. Mol. Immunol. 41, 479–493 (2004).

    Article  CAS  Google Scholar 

  57. Kramer, G., Kasper, P.T., de Jong, L. & de Koster, C.G. Quantitation of newly synthesized proteins by pulse labeling with azidohomoalanine. Methods Mol. Biol. 753, 169–181 (2011).

    Article  CAS  Google Scholar 

  58. Nicholas, H.R., Lowry, J.A., Wu, T. & Crossley, M. The Caenorhabditis elegans protein CTBP-1 defines a new group of THAP domain-containing CtBP co-repressors. J. Mol. Biol. 375, 1–11 (2008).

    Article  CAS  Google Scholar 

  59. de Bono, M. & Maricq, A.V. Neuronal substrates of complex behaviors in C. elegans. Annu. Rev. Neurosci. 28, 451–501 (2005).

    Article  CAS  Google Scholar 

  60. David, D.C. et al. Widespread protein aggregation as an inherent part of aging in C. elegans. PLoS Biol. 8, e1000450 (2010).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  62. Altun, Z.F. & Hall, D.H. Handbook of C. elegans anatomy. WormAtlas http://www.wormatlas.org/hermaphrodite/hermaphroditehomepage.htm (2012).

  63. Ghafouri, S. & McGhee, J.D. Bacterial residence time in the intestine of Caenorhabditis elegans. Nematology 9, 87–91 (2007).

    Article  Google Scholar 

  64. Haenni, S. et al. Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3′-end-seq. Nucleic Acids Res. 40, 6304–6318 (2012).

    Article  CAS  Google Scholar 

  65. Finney, M. & Ruvkun, G. The unc-86 gene product couples cell lineage and cell identity in C. elegans. Cell 63, 895–905 (1990).

    Article  CAS  Google Scholar 

  66. Duerr, J.S. Immunohistochemistry. Misc: In WormBook C. elegans Research Community 1–61 10.1895/wormbook.1.105.1 (19 June 2006).

  67. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  Google Scholar 

  68. Link, A.J. & Tirrell, D.A. Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J. Am. Chem. Soc. 125, 11164–11165 (2003).

    Article  CAS  Google Scholar 

  69. Gravato-Nobre, M.J. et al. Multiple genes affect sensitivity of Caenorhabditis elegans to the bacterial pathogen Microbacterium nematophilum. Genetics 171, 1033–1045 (2005).

    Article  CAS  Google Scholar 

  70. You, Y.J., Kim, J., Cobb, M. & Avery, L. Starvation activates MAP kinase through the muscarinic acetylcholine pathway in Caenorhabditis elegans pharynx. Cell Metab. 3, 237–245 (2006).

    Article  CAS  Google Scholar 

  71. Keith, S.A., Amrit, F.R., Ratnappan, R. & Ghazi, A. The C. elegans healthspan and stress-resistance assay toolkit. Methods 68, 476–486 (2014).

    Article  CAS  Google Scholar 

  72. Minniti, A.N. et al. Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification. Mol. Neurodegener. 4, 2 (2009).

    Article  Google Scholar 

  73. Zhang, T., Mullane, P.C., Periz, G. & Wang, J. TDP-43 neurotoxicity and protein aggregation modulated by heat shock factor and insulin/IGF-1 signaling. Hum. Mol. Genet. 20, 1952–1965 (2011).

    Article  CAS  Google Scholar 

  74. Yoneda, T. et al. Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones. J. Cell. Sci. 117, 4055–4066 (2004).

    Article  CAS  Google Scholar 

  75. Calfon, M. et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92–96 (2002).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by the Estate of Dr. Clem Jones, AO, and by grants from the Australian Research Council (DP13300101932) and the National Health and Medical Research Council of Australia (APP1037746 and APP1003150) to J.G. Mass spectrometry was undertaken at The Australian Proteome Facility, with the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy. Some strains were provided by the CGC, which is funded by the US National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440).

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M.U., V.L., Y.L.C., S.B., X.S., T.Z., H.L. and S.B. performed the experiments; M.U., V.L., Y.L.C., S.B., X.S., T.Z., H.L., S.B., M.K., H.R.N. and J.G. analyzed the data; and M.U., V.L., Y.L.C., H.R.N. and J.G. wrote the manuscript with input from all authors.

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Correspondence to Hannah R Nicholas or Jürgen Götz.

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Ullrich, M., Liang, V., Chew, Y. et al. Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans. Nat Protoc 9, 2237–2255 (2014). https://doi.org/10.1038/nprot.2014.150

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