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 expres...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 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 immunoprecipitation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.展开更多
基金supported by the National Key R&D Program of China(Grant No.2019YFA0903803)the Major Program of National Natural Science Foundation of China(Grant No.32090031)+10 种基金the General Program of National Natural Science Foundation of China(Grant Nos.31971354 and 32070610)the National Natural Science Foundation of China for Young Scholars(Grant No.32000580)the Guangdong Province Fund for Distinguished Young Scholars,China(Grant No.2021B1515020109)the Key Project from Natural Science Foundation of Guangdong Province,China(Grant No.2020B1515120034)the Guangdong Provincial Key Laboratory of Synthetic Genomics,China(Grant No.2019B030301006)the Shenzhen Key Laboratory of Synthetic Genomics,China(Grant No.ZDSYS201802061806209)the Project from Shenzhen Science and Technology Innovation Committee,China(Grant No.JCYJ20170818164014753)the Mayo Clinic Cancer Center Eagles Cancer Fund awarded to ZWthe Mayo Clinic Cancer Center Hematologic Malignancies Program awarded to ZWthe Mayo Clinic division of Hematology awarded to ZWthe Mayo Clinic Center for Biomedical Discovery awarded to SMO,United States。
文摘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 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 immunoprecipitation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
