Mass spectrometry analysis of potential interactors or RNF214
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jianping Jin
PROVIDER: PXD052393 | iProX | Sat May 18 00:00:00 BST 2024
REPOSITORIES: iProX
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Project description:We established an APEX proximity labeling strategy in coupled with mass spectrometry to identify interacting proteins of RNF214. In this approach, we first fused an engineered ascorbate peroxidase (APEX2) to either N-terminus or C-terminus of RNF214, expressed these two fusion proteins in HLF, an HCC cell line, near the endogenous level, and generated short-lived radicals around the APEX2-RNF214 fusion proteins to label biotin on nearby interactive proteins by adding hydrogen peroxide (H2O2) and biotin-phenol (also called biotin-tyramide) transiently. Biotinylated proteins were then isolated using Streptavidin resin for protein identification by mass spectrometry.
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Project description:We sought to map RBM6 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. Toward this end, we used CRISPR-cas9 methodology to establish MCF10A cell line expressing APEX2 fused to the N-terminal of RBM6, hereafter called MCF10AAPEX2-RBM6. The peroxidase activity of APEX2-RBM6 fusion was confirmed. Next, RBM6 proximal proteins that were biotinylated by APEX2 activation using hydrogen peroxidase (H2O2) in the presence of biotin-phenol were isolated and subjected to mass spectrometry. We identified 173 (P-value<0.05) RBM6 proximity-interaction partners.
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Project description:RNA binding motif protein 42 (RBM42) is an understudied gene mapped to 19q13.12 region and codes for a protein that consists of 480 amino acids, containing one RNA recognition motif (RRM) at its C-terminal region. We mapped the RBM42 interactome using ascorbate peroxidase (APEX2)-based proximity labelling combined with mass spectrometry. We utilized CRISPR-Cas9 methodology for biallelic knock-in APEX2 at the C-terminus of the endogenous RBM42 coding sequence (CDS) and established an HCT116 cell line expressing RBM42-APEX2 fusion. RBM42 interactome mapping was performed by using Control and and HCT116 expressing RBM42-APEX2 cells that were treated with DMSO or Etoposide (VP-16) that induce DNA repair and incubated in media containing biotin phenol, followed by H2O2 treatment, to activate APEX2 peroxidase activity. Pathway enrichment analysis revealed that the majority of proteins that appeared in proximity to RBM42 are implicated in mRNA splicing and processing. Furthermore, we observed a significant enrichment of proteins involved in cell cycle checkpoints and regulation, further highlighting the role of RBM42 in DNA damage response. Together, RBM42 interactome analysis substantiates its function as a splicing regulator and implicates it in additional cellular pathways related to mRNA metabolism and DNA damage response.
2023-11-22 | PXD034854 | Pride
Project description: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 of post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-APEX2 labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tehers a protein A-APEX2 (pA-APEX2) fusion protein. Activation of APEX2 labels the nearby proteins with biotin; these proteins are then purified using streptavidin beads and are identified by mass spectrometry. We demonstrate the utility of this approach by profiling the binding proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac and H4K12ac, verified the co-localization of these identified proteins with baits proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient tool to identify proteins that are proximal to modified histones.
2023-07-12 | PXD028063 | Pride
Project description:Enhanced ascorbate peroxidase 2 (APEX2) can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein, ideally within a confined compartment. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. Here we used rapamycin-dependent targeting of APEX2 to tail-anchored proteins of the endoplasmic reticulum and of the inner nuclear membrane (INM). In combination with stable isotope labeling with amino acids in cell culture (SILAC), our novel approach (rapamycin- and APEX-dependent identification of proteins by SILAC; RAPID-SILAC or RAPIDS) allowed the identification of proteins that are in close proximity to the prototypic INM-protein emerin and the vesicle-trafficking protein VAPB. In addition to well-known interaction partners, several new potential binding partners were identified. In RAPIDS, the comparison of plus/minus-rapamycin conditions allows a clear distinction between specific and non-specific hits.
2020-04-15 | PXD009783 | Pride
Project description:Enhanced ascorbate peroxidase 2 (APEX2) can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein, ideally within a confined compartment. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. Here we used rapamycin-dependent targeting of APEX2 to tail-anchored proteins of the endoplasmic reticulum and of the inner nuclear membrane (INM). In combination with stable isotope labeling with amino acids in cell culture (SILAC), our novel approach (rapamycin- and APEX-dependent identification of proteins by SILAC, or RAPIDS) allowed the identification of proteins that are in close proximity to the prototypic INM-protein emerin and the vesicle-trafficking protein VAPB. In addition to well-known interaction partners, several new potential binding partners were identified. In RAPIDS, the comparison of plus/minus-rapamycin conditions allows a clear distinction between specific and non-specific hits.
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Project description:Engineered ascorbate peroxidase APEX2 has seen widely used for spatially restricted profiling of subcellular biomolecules, but its catalytic efficiency toward newly developed probes such as biotin-aniline (Btn-An) remains suboptimal. To overcome this limitation, we performed yeast surface display based directed evolution to enhance APEX2 activity toward Btn-An. The resulting variant, L242FAPEX2, exhibits an approximately two-fold improvement in labeling efficiency, likely through enhanced enzyme-substrate interactions. This increased activity enables rapid and efficient proximity labeling while maintaining spatial specificity. After validating its performance at the ER membrane, we applied L242FAPEX2 to profile the transcriptome proximal to the midbody and identified ANLN as a previously unreported midbody-localized mRNA during telophase. Drug perturbation and reporter assays further revealed that ANLN mRNA targeting to the midbody occurs co-translationally and depends on its nascent N-terminal peptide. Together, this work establishes L242FAPEX2 as an improved and versatile tool for spatially resolved transcriptomics in complex subcellular contexts.
2026-03-26 | GSE262170 | GEO
Project description:Although we know a great deal about the subcellular location of most proteins, our knowledge of where most RNAs localize within cells remains limited. As RNA subcellular localization determines the fate, function, and regulation of both coding and non-coding RNAs , there has been substantial interest in developing new scalable approaches to study the location of RNAs in their native context. Furthermore, many locations, such as membrane-bound and membrane-less organelles, have traditionally been challenging to study due to lack of suitable tools to interrogate their constituents. Here, we describe a detailed protocol for APEX-seq that yields transcriptome-wide information of the subcellular address of RNAs that can, in principle, be applied to any subcellular location, membrane, or condensate. APEX-seq utilizes a genetically-encoded engineered ascorbate peroxidase (APEX2) tagged to a specific protein that localizes it to a region of interest. In the presence of biotin-phenol (BP) and hydrogen peroxide, APEX2 catalyzes the biotinylation of RNAs in its vicinity, which can be purified using streptavidin beads and sequenced to reveal the RNA repertoire at that subcellular location. APEX-seq experiments can be carried out by laboratory personnel trained in molecular biology. The analysis of APEX-seq data requires familiarity with standard RNA-seq workflows. With APEX2-expressing cell lines in hand, the entire procedure from labeling reaction to analysis can be completed in one week. We expect this proximity labeling approach to facilitate the unbiased discovery of RNAs localizing to different organelles and to generate hypotheses for the mechanisms and pathways involved in regulating these processes.
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Project description:Staphylococcus aureus bacteria were decorated with modified ascorbate peroxidase APEX2-YFP-CWT via the cell wall targeting (CWT) domain of lysostaphin. Cytosol of transgenic HeLa cells expressing APEX2-YFP-CWT was generated by ultracentrifugation and living bacteria were incubated in the cytosol. The bacteria were stringently washed and added to either untreated human lung microvascular endothelial cells (HuLEC) or to cells pre-treated with bacterial sphingomyelinase (β-toxin), ionomycin, or amitriptyline. After 15 min, H2O2 was added for 1 min, then the biotinylation reaction was stopped by addition Stop Solution (containing sodium azide, sodium ascorbate, and Trolox in PBS). The cells then were scraped off the substratum, centrifuged, and lysed in RIPA buffer. To remove excess biotin phenol, lysates were transferred into a 3 kDa cutoff Amicon concentrator and 5 ml RIPA buffer was added. Solution was concentrated by centrifugation to 500-1000 µL. This step was repeated once. The lysates then were incubated overnight with magnetic streptavidin beads, followed by extensive washing and eventually biotinylated proteins were eluted by addition of Laemmli buffer containing an excess of biotin.
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