title: Enhanced Purification of Ubiquitinated Proteins by Engineered Tandem Hybrid Ubiquitin-binding Domains (ThUBDs).
abstract: Ubiquitination is one of the most common post-translational modifications, regulating protein stability and function. However, the proteome-wide profiling of ubiquitinated proteins remains challenging due to their low abundance in cells. In this study, we systematically evaluated the affinity of ubiquitin-binding domains (UBDs) to different types of ubiquitin chains. By selecting UBDs with high affinity and evaluating various UBD combinations with different lengths and types, we constructed two artificial tandem hybrid UBDs (ThUBDs), including four UBDs made of DSK2p-derived ubiquitin-associated (UBA) and ubiquilin 2-derived UBA (ThUDQ2) and of DSK2p-derived UBA and RABGEF1-derived A20-ZnF (ThUDA20). ThUBD binds to ubiquitinated proteins, with markedly higher affinity than naturally occurring UBDs. Furthermore, it displays almost unbiased high affinity to all seven lysine-linked chains. Using ThUBD-based profiling with mass spectrometry, we identified 1092 and 7487 putative ubiquitinated proteins from yeast and mammalian cells, respectively, of which 362 and 1125 proteins had ubiquitin-modified sites. These results demonstrate that ThUBD is a refined and promising approach for enriching the ubiquitinated proteome while circumventing the need to overexpress tagged ubiquitin variants and use antibodies to recognize ubiquitin remnants, thus providing a readily accessible tool for the protein ubiquitination research community.
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Yuan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Yanchang
authors.initials: Y
authors.lastname: Li
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Chengpu
authors.initials: C
authors.lastname: Zhang
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Mingzhi
authors.initials: M
authors.lastname: Zhao
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Chen
authors.initials: C
authors.lastname: Deng
authors.affiliation: the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China;
authors.forename: Qiuyan
authors.initials: Q
authors.lastname: Lan
authors.affiliation: the ‖Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; and.
authors.forename: Zexian
authors.initials: Z
authors.lastname: Liu
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Na
authors.initials: N
authors.lastname: Su
authors.affiliation: the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China;
authors.forename: Jingwei
authors.initials: J
authors.lastname: Wang
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Feng
authors.initials: F
authors.lastname: Xu
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Yongru
authors.initials: Y
authors.lastname: Xu
authors.affiliation: the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China;
authors.forename: Lingyan
authors.initials: L
authors.lastname: Ping
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Lei
authors.initials: L
authors.lastname: Chang
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China;
authors.forename: Huiying
authors.initials: H
authors.lastname: Gao
authors.affiliation: the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China;
authors.forename: Junzhu
authors.initials: J
authors.lastname: Wu
authors.affiliation: the ‖Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, China; and.
authors.forename: Yu
authors.initials: Y
authors.lastname: Xue
authors.affiliation: the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China;
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: the **Departments of Structural Biology and Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 junmin.peng@stjude.org xupingghy@gmail.com.
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.affiliation: From the ‡State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, National Center for Protein Sciences Beijing, Beijing Institute of Radiation Medicine, Beijing 102206,China; the ¶School of Basic Medical Science, Key Laboratory of Combinatorial Biosynthesis and Drug Discovery of Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China; junmin.peng@stjude.org xupingghy@gmail.com.
authors.forename: Ping
authors.initials: P
authors.lastname: Xu
mesh_terms: D000818:Animals; D001665:Binding Sites; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification; D025801:Ubiquitin / Q000378:metabolism*; D057149:Ubiquitinated Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins