Journal article
Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling
Genomics, proteomics & bioinformatics, Vol.20(1), pp.87-100
02/01/2022
DOI: 10.1016/j.gpb.2021.09.003
PMCID: PMC9510856
PMID: 34555496
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.
Details
- Title: Subtitle
- Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling
- Creators
- Xinran Li - Synthetic GenomicsJiaqi Zhou - Synthetic GenomicsWenjuan Zhao - Synthetic GenomicsQing Wen - Synthetic GenomicsWeijie Wang - Synthetic GenomicsHuipai Peng - Synthetic GenomicsYuan Gao - Cold Spring Harbor LaboratoryKelly J. Bouchonville - Mayo ClinicSteven M. Offer - Mayo ClinicKuiming Chan - City University of Hong KongZhiquan Wang - Mayo ClinicNan Li - Synthetic GenomicsHaiyun Gan - Synthetic Genomics
- Resource Type
- Journal article
- Publication Details
- Genomics, proteomics & bioinformatics, Vol.20(1), pp.87-100
- Publisher
- Elsevier B.V
- DOI
- 10.1016/j.gpb.2021.09.003
- PMID
- 34555496
- PMCID
- PMC9510856
- ISSN
- 1672-0229
- eISSN
- 2210-3244
- Language
- English
- Date published
- 02/01/2022
- Academic Unit
- Pathology
- Record Identifier
- 9984618642302771
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