TY - JOUR
T1 - Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling
AU - Li, Xinran
AU - Zhou, Jiaqi
AU - Zhao, Wenjuan
AU - Wen, Qing
AU - Wang, Weijie
AU - Peng, Huipai
AU - Gao, Yuan
AU - Bouchonville, Kelly J.
AU - Offer, Steven M.
AU - Chan, Kuiming
AU - Wang, Zhiquan
AU - Li, Nan
AU - Gan, Haiyun
N1 - Publisher Copyright:
© 2022 The Authors
PY - 2022/2
Y1 - 2022/2
N2 - 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.
AB - 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.
KW - AMAPEX
KW - Modified histone
KW - Post-translationally
KW - Proximity labeling
UR - http://www.scopus.com/inward/record.url?scp=85136668754&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85136668754&partnerID=8YFLogxK
U2 - 10.1016/j.gpb.2021.09.003
DO - 10.1016/j.gpb.2021.09.003
M3 - Article
C2 - 34555496
AN - SCOPUS:85136668754
SN - 1672-0229
VL - 20
SP - 87
EP - 100
JO - Genomics, Proteomics and Bioinformatics
JF - Genomics, Proteomics and Bioinformatics
IS - 1
ER -