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.

Induction of fetal hemoglobin through enhanced translation efficiency of γ-globin mRNA

“…Next, we used the nonradioactive SUnSET method to analyze protein synthesis. 20 In this method, low doses of puromycin are incorporated into neosynthesized peptides [20][21][22] and are monitored by flow cytometry to deduce protein translation rates. The puromycin fluorescence was not significantly changed after 24 hours of Sal treatment, confirming similar levels of total protein synthesis (supplemental Figure 1C).…”

Rapidly Translated Polypeptides Are Preferred Substrates for Cotranslational Protein Degradation

“…Emetine was used to arrest elongating ribosomes to prevent ribosomal run-off. It has been shown that emetine does not interfere with the incorporation of puromycin into nascent chains (24). Total cell extracts were clarified by centrifugation at 4°C.…”

“…The reaction was terminated, and RNCs were disassembled by addition of 1 ml of urea/SDS denaturing buffer (50 mM Tris-HCl, pH 7.5, 8 M urea, 2% SDS, and 200 mM NaCl). Biotin-puromycin-labeled nascent chains were recovered by streptavidin-agarose beads, following the PUNCH approach (24). The recovery efficiency was examined by immunoblotting analysis.…”

“…SILAC-PUNCH-P Analysis-The abundance difference of individual ribosome-bound nascent chains between WT and srp1-49 cells was quantified by a combination of SILAC (stable isotope labeling by amino acids in cell culture) and PUNCH-P (puromycin-associated nascent chain proteomics) approaches (23,24). Yeast strains YXY728 and YXY730 were grown in 10 ml of synthetic complete medium containing either 30 g/ml L-lysine (light) or 31.28 g/ml L-lysine [ 13 C 6 15 N 2 ] (heavy) for at least 10 generations until the late log phase.…”

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions

“…However, this approach still provides indirect evidence for the presence of a specific sPEP, since the observed interaction of an ORF with ribosomes does not necessarily imply translation of that ORF, but it can also function in regulating the translation of other ORFs. Cutting-edge techniques such as puromycin-associated nascent chain proteomics (PUNCH-P) allowing intact ribosome-nascent polypeptide chains to be analyzed by mass spectrometry (Aviner et al, 2014) could overcome this problem, but are not yet been optimized for plants. The most direct evidence for the presence of peptides in planta can be obtained from mass spectrometry analyses.…”