PUNCH-P for global translatome profiling: Methodology, insights and comparison to other techniques.

Abstract

Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.