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Fishing for newly synthesized proteins with phosphonate-handles

Author

Listed:
  • Fleur Kleinpenning

    (Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University
    Netherlands Proteomics Centre)

  • Barbara Steigenberger

    (Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University
    Netherlands Proteomics Centre)

  • Wei Wu

    (Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University
    Netherlands Proteomics Centre)

  • Albert J. R. Heck

    (Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University
    Netherlands Proteomics Centre)

Abstract

Bioorthogonal chemistry introduces affinity-labels into biomolecules with minimal disruption to the original system and is widely applicable in a range of contexts. In proteomics, immobilized metal affinity chromatography (IMAC) enables enrichment of phosphopeptides with extreme sensitivity and selectivity. Here, we adapt and combine these superb assets in a new enrichment strategy using phosphonate-handles, which we term PhosID. In this approach, click-able phosphonate-handles are introduced into proteins via 1,3-dipolar Huisgen-cycloaddition to azido-homo-alanine (AHA) and IMAC is then used to enrich exclusively for phosphonate-labeled peptides. In interferon-gamma (IFNγ) stimulated cells, PhosID enabled the identification of a large number of IFN responsive newly synthesized proteins (NSPs) whereby we monitored the differential synthesis of these proteins over time. Collectively, these data validate the excellent performance of PhosID with efficient analysis and quantification of hundreds of NSPs by single LC-MS/MS runs. We envision PhosID as an attractive and alternative tool for studying stimuli-sensitive proteome subsets.

Suggested Citation

  • 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.
  • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-17010-0
    DOI: 10.1038/s41467-020-17010-0
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    Cited by:

    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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