Barrett Ruth M, Liu Hui-Wen, Jin Haihong, Goodman Richard H, Cohen Michael S
Vollum Institute, ‡Program in Chemical Biology, and §Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon 97210, United States.
ACS Chem Biol. 2016 Jun 17;11(6):1532-6. doi: 10.1021/acschembio.5b01076. Epub 2016 Apr 21.
Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues.
翻译调控是基因表达的一个基本组成部分，使细胞能够在没有新转录的情况下对多种刺激迅速做出反应。由于缺乏在异质组织中不同细胞群体中分析新生蛋白质组的方法，阻碍了我们在生理相关背景下对翻译调控的理解。在这里，我们描述了一种化学遗传学方法，该方法涉及通过正交酶介导将可点击的嘌呤霉素类似物掺入新生多肽中。使用这种方法，我们表明我们可以在培养的细胞和异质组织中以细胞特异性方式标记新合成的蛋白质。我们还表明，我们可以使用亲和富集和串联质谱法鉴定基因靶向细胞群体中的新生蛋白质组。我们的方法有可能为异质组织中细胞特异性翻译调控提供前所未有的见解。
