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An integrated workflow for quantitative analysis of the newly synthesized proteome

Author

Listed:
  • Toman Borteçen

    (German Cancer Research Center (DKFZ)
    Heidelberg University, Faculty of Biosciences)

  • Torsten Müller

    (Heidelberg University, Medical Faculty)

  • Jeroen Krijgsveld

    (German Cancer Research Center (DKFZ)
    Heidelberg University, Medical Faculty)

Abstract

The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.

Suggested Citation

  • 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.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-43919-3
    DOI: 10.1038/s41467-023-43919-3
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    References listed on IDEAS

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    1. 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.
    2. Fleur Kleinpenning & Barbara Steigenberger & Wei Wu & Albert J. R. Heck, 2020. "Fishing for newly synthesized proteins with phosphonate-handles," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
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