2. Academic Validation
  3. Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

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

  • Genomics Proteomics Bioinformatics. 2022 Feb;20(1):87-100. doi: 10.1016/j.gpb.2021.09.003.
Xinran Li 1,  Jiaqi Zhou 1,  Wenjuan Zhao 1,  Qing Wen 1,  Weijie Wang 1,  Huipai Peng 1,  Yuan Gao 2,  Kelly J Bouchonville 3,  Steven M Offer 4,  Kuiming Chan 5,  Zhiquan Wang 6,  Nan Li 7,  Haiyun Gan 8
Affiliations

Affiliations

  • 1 Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
  • 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
  • 3 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA.
  • 4 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA; Mayo Clinic College of Medicine, Rochester, MN 55905, USA; Mayo Clinic Cancer Center, Rochester, MN 55905, USA.
  • 5 Department of Biomedical Sciences, City University of Hong Kong, Hong Kong Special Administrative Region 999077, China; Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen 518172, China.
  • 6 Mayo Clinic College of Medicine, Rochester, MN 55905, USA; Division of Hematology, Department of Medicine, Mayo Clinic, Rochester, MN 55905, USA. Electronic address: Wang.Zhiquan@mayo.edu.
  • 7 Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Electronic address: nan.li@siat.ac.cn.
  • 8 Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Electronic address: hy.gan@siat.ac.cn.
PMID: 34555496 DOI: 10.1016/j.gpb.2021.09.003
Abstract

Proximity labeling catalyzed by promiscuous Enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To 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 protein. Activation of APEX2 labels the nearby proteins with biotin; the Biotinylated Proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.

Keywords

AMAPEX; Modified histone; Post-translationally; Proximity labeling.

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