Department of Biochemistry and Molecular Biology and ‡Department of Physics, Chemistry and Pharmacy, University of Southern Denmark , 5230 Odense, Denmark.
J Proteome Res. 2018 Jan 5;17(1):296-304. doi: 10.1021/acs.jproteome.7b00566. Epub 2017 Nov 1.
Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.
蛋白质活性可被翻译后修饰（PTMs）所调控，这是一种参与几乎所有细胞过程调控的主要分子机制。这些翻译后修饰之一是泛素化，它调节包括蛋白质降解、细胞周期、DNA 损伤修复和信号转导在内的关键过程。由于其对众多细胞功能的重要性，泛素化已成为近年来研究的热点，蛋白质组学工具极大地促进了许多泛素化靶标的鉴定。利用 StUbEx 策略将内源性泛素交换为带有表位标记的版本，我们创建了一个改良系统 StUbEx PLUS，该系统允许通过质谱精确绘制泛素化位点。将 StUbEx PLUS 应用于用蛋白酶体抑制剂处理的 U2OS 细胞，鉴定出 7762 种蛋白质上的 41589 个位点，从而揭示了这种翻译后修饰的普遍性，并证明了该方法在特定位置进行全面泛素化研究的实用性。
