Genes Dev. 27, 1834–1844 (2013)

The ability to detect rapid changes in protein synthesis under different experimental conditions can provide insights into gene expression regulation. Techniques to identify newly synthesized proteins such as MS analysis of pulse-labeled amino acids (pSILAC) and RNA sequencing of ribosome-enriched transcripts (Ribo-seq) have advanced our understanding of the kinetics of mRNA translation. However, pSILAC is not applicable to monitoring rapid changes in protein synthesis, and large-scale Ribo-seq analysis is not yet economically feasible. Aviner et al. now report a new assay called puromycin-associated nascent chain proteomics (PUNCH-P) to monitor mRNA translation. This approach quickly labels nascent polypeptides with a biotinylated form of puromycin, and a streptavidin-mediated pulldown is then subjected to MS analysis. PUNCH-P was as effective as established techniques in terms of measuring protein synthesis, and although it lacked the high resolution of Ribo-seq, PUNCH-P identified large data sets of proteins in an economically efficient manner. As a test case, the authors used this technique to identify more than 5,000 proteins that are synthesized during particular stages of the cell cycle, including known components as well as proteins that were not previously implicated in the process. For example, CCRN4L, a circadian deadenylase and PCF11, a pre-mRNA 3′ end–processing factor, were found to be upregulated during the S phase and M phase, respectively. Finally, the authors demonstrated the utility of the method to assay mRNA translation in tissues by successfully labeling newly synthesized proteins in a mouse brain. Taken together, PUNCH-P may be a cost-effective approach to measure rapid changes in global protein synthesis in cells and tissues.