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Genome-wide identification and quantification of protein synthesis in cultured cells and whole tissues by puromycin-associated nascent chain proteomics (PUNCH-P)

Nature Protocols volume 9, pages 751–760 (2014)Cite this article

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Abstract

Regulation of mRNA translation has a pivotal role in modulating protein levels, and the genome-wide identification of proteins synthesized at a given time is indispensable to our understanding of gene expression. This protocol describes the mass-spectrometric analysis of newly synthesized proteins from cultured cells or whole tissues by using a biotinylated derivative of puromycin, which becomes incorporated into nascent polypeptide chains by ribosome catalysis. In this method, termed puromycin-associated nascent chain proteomics (PUNCH-P), intact ribosome-nascent chain complexes are first recovered from cells by ultracentrifugation, followed by biotin-puromycin labeling of newly synthesized proteins, streptavidin affinity purification and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Unlike methods that require in vivo labeling, the sensitivity and coverage of PUNCH-P depend only on the amount of starting material and not on the duration of labeling, thus enabling the measurement of rapid fluctuations in protein synthesis. The protocol requires 3 d for sample preparation and analysis.

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Figure 1: The PUNCH-P strategy for monitoring protein synthesis.
Figure 2: Western blot analysis of biotin-puromycin-labeled proteins and optimization of biotin-puromycin and affinity resin amounts.
Figure 3: Hierarchical clustering of proteomic output.

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Acknowledgements

O.E.-S. acknowledges support from the Israel Science Foundation (grant 1036/12) and The Legacy Heritage Bio-Medical Program of the Israel Science Foundation (grant no. 1629/13). T.G. acknowledges support from the Israel Science Foundation (grant 1617/12) and the Israeli Centers of Research Excellence (I-CORE), Gene Regulation in Complex Human Disease, Center no. 41/11.

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Author notes

  1. Tamar Geiger and Orna Elroy-Stein: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel

    Ranen Aviner & Orna Elroy-Stein

  2. Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Tamar Geiger

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  1. Ranen Aviner
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Contributions

R.A., T.G. and O.E.-S. designed the experiments. R.A. performed the biochemical experiments. T.G. performed the mass spectrometry experiments and data analysis. R.A., T.G. and O.E.-S. wrote the paper.

Corresponding author

Correspondence to Tamar Geiger.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of puromycin and biotin-puromycin labeling efficiencies under different conditions.

Intact cultured HeLa cells, lysed cells or isolated polysomes (denoted ‘In-culture’, ‘In-lysate’ or ‘On-polysome’, respectively) were treated with 1μM puromycin (Puro) or 1 μM biotin-puromycin (Biot-PU) for 30 min at 37 °C or left untreated. 20 μg of total protein from each sample were resolved on a 12% SDS-PAGE; the membrane was stained with Ponceau S as loading control and probed sequentially with a puromycin antibody (clone 12D10) and streptavidin-HRP. Note that the puromycin antibody detects only underivatized puromycin and not biotin-puromycin, probably due to epitope masking by biotin. The dashed red box delineates the advantage of ribosome purification for high labeling efficiency using biotin-puromycin.

Supplementary Figure 2 Densitometry quantification related to Figure 2.

(a) Quantification of immunoblot in Figure 2b. (b) Quantification of immunoblot in Figure 2C.

Supplementary information

Supplementary Figure 1

Comparison of puromycin and biotin-puromycin labeling efficiencies under different conditions.. (PDF 252 kb)

Supplementary Figure 2

Densitometry quantification related to Figure 2. (PDF 186 kb)

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Aviner, R., Geiger, T. & Elroy-Stein, O. Genome-wide identification and quantification of protein synthesis in cultured cells and whole tissues by puromycin-associated nascent chain proteomics (PUNCH-P). Nat Protoc 9, 751–760 (2014). https://doi.org/10.1038/nprot.2014.051

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