Quantitative nascent proteome profiling by dual-pulse labelling with O-propargyl-puromycin and stable...

Monitoring translational regulation in response to environmental signals is crucial for understanding cellular proteostasis. However, only limited approaches are currently available for quantifying acute changes in protein synthesis induced by stimuli. Recently, a clickable puromycin analogue, O-propargyl-puromycin (OPP), was developed and applied to label the C-termini of nascent polypeptide chains (NPCs). Following affinity purification via a click reaction, OPP allows for a proteomic analysis of NPCs. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from significant non-specific protein binding, which hinders accurate quantification of the nascent proteins. To address this issue, we employed dual-pulse labelling of NPCs with both OPP and stable isotope-labelled amino acids to distinguish bona fide NPCs from non-specific proteins, thereby enabling the accurate quantitative profiling of NPCs. We applied this method to dissecting translation responses upon transcriptional inhibition and quantified ∼3,000 nascent proteins. We found that the translation of a subset of ribosomal proteins (e.g. RPSA, RPLP0) as well as signalling proteins (e.g. BCAR3, EFNA1, DUSP1) was significantly repressed by transcription inhibition. Together, the present method provides an accurate and broadly applicable nascent proteome profiling for many biological applications at the level of translation.