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Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Genomics, Proteomics & Bioinformatics 2022年第1期20卷 87-100页

作者： Xinran Li Jiaqi Zhou Wenjuan Zhao Qing Wen Weijie Wang Huipai Peng Yuan Gao Kelly J.Bouchonville Steven M.Offer Kuiming Chan Zhiquan Wang Nan Li Haiyun Gan Shenzhen Key Laboratory of Synthetic Genomics Guangdong Provincial Key Laboratory of Synthetic GenomicsCAS Key Laboratory of Quantitative Engineering BiologyShenzhen Institute of Synthetic BiologyShenzhen Institute of Advanced TechnologyChinese Academy of SciencesShenzhen 518055China Cold Spring Harbor Laboratory Cold Spring HarborNY 11724USA Department of Molecular Pharmacology and Experimental Therapeutics Mayo ClinicRochesterMN 55905USA Mayo Clinic College of Medicine RochesterMN 55905USA Mayo Clinic Cancer Center RochesterMN 55905USA Department of Biomedical Sciences City University of Hong KongHong Kong Special Administrative Region 999077China Key Laboratory of Biochip Technology Biotech and Health CentreShenzhen Research Institute of City University of Hong KongShenzhen 518172China Division of Hematology Department of MedicineMayo ClinicRochesterMN 55905USA

Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein ***,current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified *** address this limitation,we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2(pA-APEX2) labeling(amapex).In this method,a modified protein is bound in situ by a specific antibody,which then tethers a pA-APEX2 fusion *** of APEX2 labels the nearby proteins with biotin;the biotinylated proteins are then purified using streptavidin beads and identified by mass *** demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3 K27 me3,H3 K9 me3,H3 K4 me3,H4 K5 ac,and H4 K12 ac,as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome ***,amapex is an efficient method to identify proteins that are proximal to modified histones.

关键词： Proximity labeling Post-translationally amapex Modified histone

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