Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins.
Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)-binding proteins. We report diazirine-based photoaffinity probes that can capture Ub-binding proteins in cell lysates, and show that diazirines are preferable to aryl azides as the photo-crosslinking group, since they decrease non-selective capture. Photoaffinity probes containing at least two Ub units were required to effectively capture Ub-binding proteins. Different capture selectivity was observed for probes containing diubiquitin moieties with different types of linkages, thus indicating the potential to develop linkage-dependent probes for selectively profiling Ub-binding proteins under various cellular conditions.
Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and University of Science and Technology of China, Hefei, 230026, China.
Jun
J
Liang
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Lin
L
Zhang
Department of Chemistry, Tsinghua University, Beijing, 100084, China.
Xiang-Long
XL
Tan
Department of Chemistry, Tsinghua University, Beijing, 100084, China.
Yun-Kun
YK
Qi
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Shan
S
Feng
MOE Key Laboratory of Bioinformatics, Tsinghua-Peking Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Haiteng
H
Deng
Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and University of Science and Technology of China, Hefei, 230026, China.
Yijing
Y
Yan
Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and University of Science and Technology of China, Hefei, 230026, China.
Ji-Shen
JS
Zheng
Department of Chemistry, Tsinghua University, Beijing, 100084, China.
Lei
L
Liu
Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and University of Science and Technology of China, Hefei, 230026, China.
Chang-Lin
CL
Tian
D003978:Diazomethane / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000737:chemistry; D014452:Ubiquitins / Q000737:chemistry / Q000302:isolation & purification*
D019920:Photoaffinity Labels; C109184:UBD protein, human; D014452:Ubiquitins; D003978:Diazomethane
native chemical ligation; photoaffinity labelling; protein modifications; protein synthesis; ubiquitination