Jeon Hong Bae, Choi Eun Soo, Yoon Jong Hyuk, Hwang Jin Ha, Chang Jong Wook, Lee Eun Kyung, Choi Hyun Woo, Park Zee-Yong, Yoo Yung Joon
Department of Life Science, Gwangju Institute of Science & Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Republic of Korea.
Biochem Biophys Res Commun. 2007 Jun 8;357(3):731-6. doi: 10.1016/j.bbrc.2007.04.015. Epub 2007 Apr 12.
There is a growing need for the large-scale identification of the ubiquitinated proteins and ubiquitin attachment sites. As part of this effort, we generated a transgenic mouse expressing a tagged ubiquitin in the heart. We found that the majority of ubiquitinated proteins in mouse heart are insoluble in detergent-free buffer and were chemically cleaved after methionine with CNBr. CNBr cleaved the proteins into smaller polypeptides while preserving the ubiquitin chains. Ubiquitin-conjugated polypeptides were then purified under denaturing conditions, digested with Lys-C and trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. We identified 121 proteins that were ubiquitinated in mouse heart, and we detected 33 ubiquitination sites in 21 of the proteins. Components of cardiac muscle and many mitochondrial proteins were identified as substrates for ubiquitination, strongly suggesting that proteins related to major heart functions such as contraction and energy production are under continuous quality control by the ubiquitin system.
对泛素化蛋白和泛素连接位点进行大规模鉴定的需求日益增长。作为这项工作的一部分，我们构建了在心脏中表达带标签泛素的转基因小鼠。我们发现，小鼠心脏中的大多数泛素化蛋白不溶于无去污剂缓冲液，并在蛋氨酸后用溴化氰（CNBr）进行化学切割。CNBr将蛋白切割成较小的多肽，同时保留泛素链。然后在变性条件下纯化泛素缀合的多肽，用Lys-C和胰蛋白酶消化，并通过液相色谱 - 串联质谱分析。我们鉴定出121种在小鼠心脏中被泛素化的蛋白，并在其中21种蛋白中检测到33个泛素化位点。心肌成分和许多线粒体蛋白被鉴定为泛素化的底物，强烈表明与心脏主要功能如收缩和能量产生相关的蛋白处于泛素系统的持续质量控制之下。
