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 analog, 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 labeling of NPCs with both OPP and stable isotope labeled 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 signaling 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.

中文翻译：

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通过使用O-炔丙基-嘌呤霉素和稳定的同位素标记的氨基酸进行双脉冲标记，对新生蛋白质组进行定量分析。

监测响应于环境信号的翻译调节对于理解细胞蛋白稳态至关重要。但是，目前只有有限的方法可用于量化由刺激诱导的蛋白质合成的急性变化。最近，可点击的嘌呤霉素类似物O-炔丙基嘌呤霉素（OPP）已开发并应用于标记新生多肽链（NPC）的C末端。通过点击反应进行亲和纯化后，OPP可以对NPC进行蛋白质组学分析。尽管有其优势，但使用磁珠或树脂对NPC进行亲和纯化会固有地遭受显着的非特异性蛋白质结合，这阻碍了对新生蛋白质的准确定量。为了解决这个问题，我们采用NPC的双脉冲标记同时使用OPP和稳定同位素标记的氨基酸来区分善意来自非特异性蛋白质的NPC，从而能够对NPC进行准确的定量分析。我们将这种方法应用于在转录抑制后解析翻译反应并量化了约3,000个新生蛋白质。我们发现，转录抑制显着抑制了核糖体蛋白（例如RPSA，RPLP0）以及信号蛋白（例如BCAR3，EFNA1，DUSP1）的子集的翻译。总之，本方法为翻译水平的许多生物学应用提供了准确且广泛适用的新生蛋白质组图谱。