Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation.
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.
Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.
Ranen
R
Aviner
Tamar
T
Geiger
Orna
O
Elroy-Stein
D000818:Animals; D001921:Brain / Q000378:metabolism; D002453:Cell Cycle / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012333:RNA, Messenger / Q000378:metabolism*; D015203:Reproducibility of Results; D012270:Ribosomes / Q000378:metabolism
D011506:Proteins; D012333:RNA, Messenger
PUNCH-P; cell cycle; protein synthesis; proteomics; puromycin; translation