title: Genome-wide identification and quantification of protein synthesis in cultured cells and whole tissues by puromycin-associated nascent chain proteomics (PUNCH-P).
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.
authors.affiliation: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Ranen
authors.initials: R
authors.lastname: Aviner
authors.affiliation: 1] Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. [2].
authors.forename: Tamar
authors.initials: T
authors.lastname: Geiger
authors.affiliation: 1] Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel. [2].
authors.forename: Orna
authors.initials: O
authors.lastname: Elroy-Stein
mesh_terms: D001710:Biotin / Q000737:chemistry; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D014176:Protein Biosynthesis / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D011691:Puromycin / Q000737:chemistry*; D012270:Ribosomes / Q000235:genetics; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014461:Ultracentrifugation
chemical_list: D011691:Puromycin; D001710:Biotin