Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation |
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Authors: | Ranen Aviner Tamar Geiger Orna Elroy-Stein |
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Affiliation: | 1.Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel;;2.Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel |
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Abstract: | Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions. |
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Keywords: | cell cycle proteomics translation protein synthesis puromycin PUNCH-P |
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