Uchiyama Junki, Roy Rohini, Wang Dan Ohtan, Morikawa Kazuya, Kawahara Yuka, Iwasaki Mio, Yoshino Chiaki, Mishima Yuichiro, Ishihama Yasushi, Imami Koshi
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, 606-8501, Japan.
iScience. 2022 Jun 3;25(7):104516. doi: 10.1016/j.isci.2022.104516. eCollection 2022 Jul 15.
Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.
细胞整体翻译通常使用核糖体分析或定量质谱法进行测量，但这些方法无法在新生多肽链（NPC）延伸水平及相关共翻译事件层面提供直接信息。在此，我们描述了pSNAP，这是一种通过亲和富集嘌呤霉素和稳定同位素标记多肽对NPC进行全蛋白质组分析的方法。pSNAP不需要核糖体纯化和/或化学标记，并且能够捕获具有蛋白质N端偏向位置特征的NPC。我们应用pSNAP评估了西咪替塞替（一种潜在的癌症分子疗法）的效果，并揭示了通过酪蛋白激酶II和mTOR途径的急性翻译抑制。我们还对NPC上的修饰进行了表征，并证明不同类型修饰（如组蛋白H1.5 N端区域的乙酰化和磷酸化）的组合可以调节与核糖体相关因子的相互作用。因此，pSNAP为在全蛋白质组范围内剖析共翻译调控提供了一个框架。
