非经典（K6, K11, K27, K29, K33等）泛素化近期被发现在多种细胞内生物过程中发挥关键调控作用。然而非经典泛素化修饰蛋白的获取困难限制了其生物化学等研究的开展。本项目拟通过发展方法实现非经典泛素修饰蛋白、泛素链探针的高效率合成，力求从根本上解决蛋白获取的难题，并将获取的非经典泛素修饰蛋白用于研究其涉及的动态修饰调控，阐明其生物化学过程及生物物理机制。具体计划是：①发展异泛素及溴带辅基方法，实现非经典泛素链及修饰蛋白的高效化学合成；②发展光亲和二泛素探针及二硫键交联的活性二泛素探针，高效实现特异性去泛素化酶的捕获、筛查及泛素链与泛素识别酶、去修饰酶复合物的构建；③阐明非经典K27泛素链的特异性生成及水解机制；④解析蛋白酶体特异性识别分叉泛素链及其去泛素化分子机制。本项目的实施将从方法创新的视角大幅提升非经典泛素研究的能力，为国内外研究提供实用的化学生物学方法及蛋白质探针工具。

Atypical ubiquitination（K6, K11, K27, K29, K33 etc.）has recently been revealed to play key regulatory roles in a variety of intracellular biological processes. However, the difficulty of obtaining atypical ubiquitin chains and ubiquitinated proteins limits the development of their biochemistry and biophysical studies. This project plans to carry out systematic studies on the development of new strategies for the efficient chemical synthesis of atypical ubiquitinated proteins and ubiquitin probes. These valuable ubiquitin reagents will be used to elucidate the biochemical and biophysical mechanisms of atypical protein ubiquitination. Specifically, we plan to examine the following ideas: 1) develop "isoUb" unit and alkyl bromides based "auxiliary handle" to acheive the efficient chemical synthesis of atypical ubiquitin chains and ubiquitinated proteins; 2) develop novel diubiquitin-based photoaffinity probes and disulfide cross-linked active probes for profiling, identification of linkage specific deubiquitinases, as well as to assemble the complex of atypical ubiquitin chains and their readers, writers or erasers; 3) elucidate the specific generation and hydrolysis mechanism of atypical K27 linked ubiquitin chains; 4) examine the mechanism of proteasome-specific identification of branched ubiquitin chains and the molecular mechanism of proteasomal degradation. The implementation of this project will enhance the study of atypical protein ubiquitination from the perspective of method innovation, as well as provide practical chemical biology methods and protein probe toolbox for further research.