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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.

Change history

  • 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.

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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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