1.Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling
Li XINRAN ; Zhou JIAQI ; Zhao WENJUAN ; Wen QING ; Wang WEIJIE ; Peng HUIPAI ; Gao YUAN ; J.Bouchonville KELLY ; M.Offer STEVEN ; Chan KUIMING ; Wang ZHIQUAN ; Li NAN ; Gan HAIYUN
Genomics, Proteomics & Bioinformatics 2022;20(1):87-100
Proximity labeling catalyzed by promiscuous enzymes,such as APEX2,has emerged as a powerful approach to characterize multiprotein complexes and protein-protein interactions.How-ever,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 iden-tified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipi-tation.Overall,AMAPEX is an efficient method to identify proteins that are proximal to modified histones.
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