Quantitative nascent proteome profiling by pulse labeling of O-propargyl-puromycin and stable isotope labeled amino acids

Recently, a clickable puromycin analog, O-propargyl-puromycin (OPP), was developed and applied to label C-termini of nascent polypeptide chains (NPCs), followed by a proteomic analysis of NPCs that can be affinity purified via click reaction. Despite its advantage, the affinity purification of NPCs using magnetic beads or resins inherently suffers from dozens of non-specific protein binding, which results in hindering accurate quantification of the nascent proteins. To address this issue, we performed dual pulse labeling of NPCs with both OPP and stable isotope labeled amino acids that allows us to distinguish bona fide NPCs from non-specific proteins, thereby enabling accurate quantitative profiling of NPCs.