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. 2017 Jul 20;7(14):e2397.
doi: 10.21769/BioProtoc.2397.

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Ines Erdmann  1   2 , Kathrin Marter  1   2   3 , Oliver Kobler  4 , Sven Niehues  5   6 , Julia Bussmann  5   6 , Anke Müller  1   2 , Julia Abele  1   2 , Erik Storkebaum  5   6 , Ulrich Thomas  4 , Daniela C Dieterich  1   2   3
Affiliations

Cell Type-specific Metabolic Labeling of Proteins with Azidonorleucine in Drosophila

Ines Erdmann et al. Bio Protoc. .

Abstract

Advanced mass spectrometry technology has pushed proteomic analyses to the forefront of biological and biomedical research. Limitations of proteomic approaches now often remain with sample preparations rather than with the sensitivity of protein detection. However, deciphering proteomes and their context-dependent dynamics in subgroups of tissue-embedded cells still poses a challenge, which we meet with a detailed version of our recently established protocol for cell-selective and temporally controllable metabolic labeling of proteins in Drosophila. This method is based on targeted expression of a mutated variant of methionyl-tRNA-synthetase, MetRSL262G, which allows for charging methionine tRNAs with the non-canonical amino acid azidonorleucine (ANL) and, thus, for detectable ANL incorporation into nascent polypeptide chains.

Keywords: Click chemistry; Drosophila melanogaster; Metabolic labeling; Protein synthesis; Proteomic profiling.

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Figures

Figure 1.
Figure 1.. Preparation of ANL-containing fly food medium.
A. Ingredients for ONM are shown: R (mashed raisins); S (semolina); BS (sugar beet syrup); H (honey). B. Semolina and Agar-Agar are added to water and allowed to swell, SAA. C. Raisins and Baker’s yeast (Y), sugar beet syrup (BS), and honey (H), are added into water and boiled for 5 min. D. Afterwards, the SAA mixture is added and boiled once more. After cooling down to 50 °C with stirring from time to time, Nigapin and ANL are added and mixed thoroughly. E and F. Media is then aliquoted and allowed to cool down completely before storage at 4 °C.
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References

    1. Andersen J. S., Lam Y. W., Leung A. K., Ong S. E., Lyon C. E., Lamond A. I. and Mann M.(2005). Nucleolar proteome dynamics. Nature 433(7021): 77-83. - PubMed
    1. Beatty K. E., Liu J. C., Xie F., Dieterich D. C., Schuman E. M., Wang Q. and Tirrell D. A.(2006). Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew Chem Int Ed Engl 45(44): 7364-7367. - PubMed
    1. Bellen H. J. and Budnik V.(2000). Drosophila, a laboratory manual. In: Ashburner, M., Hawley, S. and Sullivan. B(Eds). Cold Spring Harbor Laboratory. chap. 11.
    1. Dieterich D. C., Hodas J. J., Gouzer G., Shadrin I. Y., Ngo J. T., Triller A., Tirrell D. A. and Schuman E. M.(2010). In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons . Nat Neurosci 13(7): 897-905. - PMC - PubMed
    1. Dieterich D. C., Link A. J., Graumann J., Tirrell D. A. and Schuman E. M.(2006). Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging(BONCAT). Proc Natl Acad Sci U S A 103(25): 9482-9487. - PMC - PubMed

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