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Stable-isotope labeling with amino acids in nematodes

Abstract

We describe an approach for accurate quantitation of global protein dynamics in Caenorhabditis elegans. We adapted stable-isotope labeling with amino acids in cell culture (SILAC) for nematodes by feeding worms a heavy lysine– and heavy arginine–labeled Escherichia coli strain and report a genetic solution to elminate the problem of arginine-to-proline conversion. Combining our approach with quantitative proteomics methods, we characterized the heat-shock response in worms.

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Figure 1: Elimination of arginine-to-proline conversion using orn-1–targeted RNAi via feeding facilitates stable-isotope labeling with amino acids.
Figure 2: SILAC in nematodes, taking the analysis of the heat-shock response as an example.
Figure 3: Analysis of C. elegans heat-shock response using SILAC in nematodes.

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Acknowledgements

This work was supported by grants from the Wellcome Trust (083524/Z/07/Z, 073980/Z/03/Z, 08136/Z/03/Z and 0909444/Z/09/Z) and by the EU FP7 Prospects network (HEALTH-F4-2008-201648). A.I.L. is funded as a Wellcome Trust Principal Research fellow and A.G. as a Senior Wellcome Trust Senior Research fellow. S.C. is supported by a Royal Society of Edinburgh fellowship. D.P.X. is funded as an Association for International Cancer Research fellow. We thank T. Palmer (University of Dundee) for providing the Keio library, F. Sargent for helpful discussions and F. Wheatley for her help.

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Authors and Affiliations

Authors

Contributions

A.P.B. and E.P. cloned, passaged and treated all C. elegans samples. M.L. performed all protein analysis. M.L., A.P.B., A.G. and A.I.L. wrote the paper. A.P.B., R.T.H., G.B. and S.C. generated the E. coli auxotroph strains. D.P.X., A.G. and A.I.L. provided mentorship and financed the project.

Corresponding authors

Correspondence to Anton Gartner or Angus I Lamond.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 2 (PDF 731 kb)

Supplementary Table 1

MaxQuant protein output for stable-isotope labeling with amino acids analysis. (XLSX 1783 kb)

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Larance, M., Bailly, A., Pourkarimi, E. et al. Stable-isotope labeling with amino acids in nematodes. Nat Methods 8, 849–851 (2011). https://doi.org/10.1038/nmeth.1679

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