期刊
GENOMICS PROTEOMICS & BIOINFORMATICS
卷 20, 期 1, 页码 87-100出版社
ELSEVIER
DOI: 10.1016/j.gpb.2021.09.003
关键词
Proximity labeling; Post-translationally; AMAPEX; Modified histone
资金
- National Key R&D Program of China [2019YFA0903803]
- Major Program of National Natural Science Foundation of China [32090031]
- General Program of National Natural Science Foundation of China [31971354, 32070610]
- National Natural Science Foundation of China for Young Scholars [32000580]
- Guangdong Province Fund for Distinguished Young Scholars, China [2021B1515020109]
- Natural Science Foundation of Guangdong Province, China [2020B1515120034]
- Guangdong Provincial Key Laboratory of Synthetic Genomics, China [2019B030301006]
- Shenzhen Key Laboratory of Synthetic Genomics, China [ZDSYS201802061806209]
- Shenzhen Science and Technology Innovation Committee, China [JCYJ20170818164014753]
- Mayo Clinic Cancer Center Eagles Cancer Fund
- Mayo Clinic Cancer Center Hematologic Malignancies Program
- Mayo Clinic division of Hematology
- Mayo Clinic Center for Biomedical Discovery
AMAPEX is a new method that identifies proteins near modified histones using antibody-mediated protein labeling, overcoming limitations of current approaches. This method utilizes biotin labeling to mark nearby proteins, which are then identified by mass spectrometry, demonstrating its utility in studying proximal protein modifications.
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.
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