Dan Songsong, Kang Bo, Duan Xiaotao, Wang Ying-Jie
State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou, Zhejiang 310003, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, Zhejiang 310003, China.
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Biochem Biophys Res Commun. 2015 Jan 16;456(3):714-20. doi: 10.1016/j.bbrc.2014.12.043. Epub 2014 Dec 15.
The octamer-binding transcription factor 4 (Oct4) is essential for maintaining the self-renewal and pluripotency of embryonic stem cells (ESCs). Post-translational modifications (PTMs) of Oct4 critically control its structure, function and intracellular localization. However, determination of Oct4 PTM profiles has largely been restricted by the quantity and purity of the Oct4 protein samples required for mass spectrometric analyses. In this study, by incubating the Escherichia coli-derived His-tagged Oct4 proteins with the whole cell lysates of a variety of human cells followed by retrieving the reacted Oct4 proteins with the Ni-NTA beads, we developed a labor- and cost-effective in vitro PTM method that allowed for mass spectrometric determination of the phosphorylation profiles of Oct4 proteins exposed to various cell-free systems. A number of Oct4 phosphorylation sites that were commonly present in all the cell-free systems or specifically present in a particular cellular context were identified, indicating that Oct4 is controlled by both common and distinct PTM regulatory pathways. Our work provided a proof-of-concept that such a cell-free system-based in vitro PTM approach can be applied to systematically map out the physiologically-relevant PTM sites in Oct4 proteins, which opened up an avenue to fully decipher the Oct4 PTM barcodes in various cellular contexts.
八聚体结合转录因子4（Oct4）对于维持胚胎干细胞（ESC）的自我更新和多能性至关重要。Oct4的翻译后修饰（PTM）严格控制其结构、功能和细胞内定位。然而，Oct4 PTM谱的确定在很大程度上受到质谱分析所需的Oct4蛋白质样品的数量和纯度的限制。在本研究中，通过将大肠杆菌衍生的His标签Oct4蛋白与多种人类细胞的全细胞裂解物孵育，然后用Ni-NTA磁珠回收反应后的Oct4蛋白，我们开发了一种经济高效的体外PTM方法，该方法允许通过质谱法测定暴露于各种无细胞系统的Oct4蛋白的磷酸化谱。鉴定出了许多在所有无细胞系统中普遍存在或在特定细胞环境中特异性存在的Oct4磷酸化位点，表明Oct4受常见和独特的PTM调节途径的控制。我们的工作提供了一个概念验证，即这种基于无细胞系统的体外PTM方法可用于系统地绘制Oct4蛋白中与生理相关的PTM位点，这为在各种细胞环境中全面解读Oct4 PTM条形码开辟了一条途径。
