Li, Xinran, Zhou, Jiaqi, Zhao, Wenjuan, Wen, Qing, Wang, Weijie, Peng, Huipai, Gao, Yuan, Bouchonville, Kelly J, Offer, Steven M, Chan, Kuiming, Wang, Zhiquan, Li, Nan, Gan, Haiyun (September 2021) Defining Proximity Proteomics of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling. Genomics Proteomics and Bioinformatics. ISSN 1672-0229
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 (APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a protein A-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and are 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 tool to identify proteins that are proximal to modified histones.
| Item Type: | Paper |
|---|---|
| Subjects: | Investigative techniques and equipment > assays bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > histone bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein methylation > histone methylation |
| CSHL Authors: | |
| Communities: | CSHL labs > Vakoc lab |
| SWORD Depositor: | CSHL Elements |
| Depositing User: | CSHL Elements |
| Date: | 20 September 2021 |
| Date Deposited: | 30 Sep 2021 17:34 |
| Last Modified: | 07 Oct 2022 00:56 |
| PMCID: | PMC9510856 |
| URI: | https://repository.cshl.edu/id/eprint/40374 |
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