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THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog

Author

Listed:
  • Bob J. Ignacio

    (Radboud University)

  • Jelmer Dijkstra

    (Radboud University
    The Netherlands Cancer Institute)

  • Natalia Mora

    (Radboud University)

  • Erik F. J. Slot

    (Radboud University)

  • Margot J. Weijsten

    (Radboud University)

  • Erik Storkebaum

    (Radboud University)

  • Michiel Vermeulen

    (Radboud University
    The Netherlands Cancer Institute)

  • Kimberly M. Bonger

    (Radboud University)

Abstract

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog β-ethynylserine (βES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding βES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.

Suggested Citation

  • Bob J. Ignacio & Jelmer Dijkstra & Natalia Mora & Erik F. J. Slot & Margot J. Weijsten & Erik Storkebaum & Michiel Vermeulen & Kimberly M. Bonger, 2023. "THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-39063-7
    DOI: 10.1038/s41467-023-39063-7
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    References listed on IDEAS

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    1. Sven Niehues & Julia Bussmann & Georg Steffes & Ines Erdmann & Caroline Köhrer & Litao Sun & Marina Wagner & Kerstin Schäfer & Guangxia Wang & Sophia N. Koerdt & Morgane Stum & Sumit Jaiswal & Uttam L, 2015. "Impaired protein translation in Drosophila models for Charcot–Marie–Tooth neuropathy caused by mutant tRNA synthetases," Nature Communications, Nature, vol. 6(1), pages 1-13, November.
    2. J. A. Marchand & M. E. Neugebauer & M. C. Ing & C.-I. Lin & J. G. Pelton & M. C. Y. Chang, 2019. "Discovery of a pathway for terminal-alkyne amino acid biosynthesis," Nature, Nature, vol. 567(7748), pages 420-424, March.
    3. Ines Erdmann & Kathrin Marter & Oliver Kobler & Sven Niehues & Julia Abele & Anke Müller & Julia Bussmann & Erik Storkebaum & Tamar Ziv & Ulrich Thomas & Daniela C. Dieterich, 2015. "Cell-selective labelling of proteomes in Drosophila melanogaster," Nature Communications, Nature, vol. 6(1), pages 1-11, November.
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    1. Toman Borteçen & Torsten Müller & Jeroen Krijgsveld, 2023. "An integrated workflow for quantitative analysis of the newly synthesized proteome," Nature Communications, Nature, vol. 14(1), pages 1-16, December.

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