期刊
NATURE PROTOCOLS
卷 13, 期 3, 页码 530-550出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.147
关键词
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资金
- Hundred Talents Program C of the Chinese Academy of Sciences [2017-045]
- Guangdong Science and Technology Projects [2014B050504008, 2014B050502012, 2014B020225002, 2014B030301058]
- NWO
- ERC grant Ubicode [281699]
- EMBL Interdisciplinary Postdoc (EIPOD) Programme under a grant from the Marie Sklodowska-Curie Actions COFUND [664726]
- European Research Council (ERC) [281699] Funding Source: European Research Council (ERC)
biquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment-mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTPTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LCLC)-MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.
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