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.