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Proximity biotinylation of Expi293F cells overexpressing EGFR by EGFR-FabID

ABSTRACT: Expi293F cells were transfected with pcDNA3.1-EGFR and treated with AGIA-FabID or EGFR-FabID in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD039449 | JPOST Repository | Tue Nov 14 00:00:00 GMT 2023

REPOSITORIES: jPOST

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			Action	DRS
	158_Yamada_19_293F_EGFR_OE_AGIA_FabID_n1_FusionIT.raw	Raw
	160_Yamada_22_293F_EGFR_OE_EGFR_FabID_n1_FusionIT.raw	Raw
	180_Yamada_20_293F_EGFR_OE_AGIA_FabID_n2_Tama_FusionIT.raw	Raw
	182_Yamada_23_293F_EGFR_OE_EGFR_FabID_n2_Tama_FusionIT.raw	Raw
	184_Yamada_21_293F_EGFR_OE_AGIA_FabID_n3_Tama_FusionIT.raw	Raw

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Publications

Proximity extracellular protein-protein interaction analysis of EGFR using AirID-conjugated fragment of antigen binding.

Yamada Kohdai K Shioya Ryouhei R Nishino Kohei K Furihata Hirotake H Hijikata Atsushi A Kaneko Mika K MK Kato Yukinari Y Shirai Tsuyoshi T Kosako Hidetaka H Sawasaki Tatsuya T

Nature communications 20231214 1

Receptor proteins, such as epidermal growth factor receptor (EGFR), interact with other proteins in the extracellular region of the cell membrane to drive intracellular signalling. Therefore, analysis of extracellular protein-protein interactions (exPPIs) is important for understanding the biological function of receptor proteins. Here, we present an approach using a proximity biotinylation enzyme (AirID) fusion fragment of antigen binding (FabID) to analyse the proximity exPPIs of EGFR. AirID w ...[more]

PMID: 38097606

Similar Datasets

Proximity biotinylation of Expi293F cells stably expressing EGFR by EGFR-FabID

Project description:Expi293F cells stably expressing EGFR were treated with AGIA-FabID or EGFR-FabID in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043525 | JPOST Repository

Proximity biotinylation of A431 cells by EGFR-FabID

Project description:A431 cells were pretreated with DMSO or gefitinib and unstimulated or stimulated with EGF. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD039450 | JPOST Repository

Proximity biotinylation of NCI-H226 cells by EGFR-FabID

Project description:NCI-H226 cells were pretreated with DMSO or gefitinib and unstimulated or stimulated with EGF. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD039451 | JPOST Repository

Proximity biotinylation of A431 cells with gefitinib alone treatment by EGFR-FabID

Project description:A431cells were pretreated with DMSO or gefitinib. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043526 | JPOST Repository

Proximity biotinylation of NCI-H226 cells with gefitinib alone treatment by EGFR-FabID

Project description:NCI-H226 cells were pretreated with DMSO or gefitinib. Then AGIA-FabID or EGFR-FabID were added to the cells in three biological replicates. Cell lysates were subjected to methanol-chloroform precipitation and digested with trypsin. Biotinylated peptides were purified by Tamavidin 2-REV and identified by LC-MS/MS analysis.

2023-11-14 | PXD043527 | JPOST Repository

DNA Modifications Enabling Proximity Biotinylation.

Project description:Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

| S-EPMC10119920 | biostudies-literature

Identification ubiquitination of EGFR by LC-MS/MS

Project description:Previously, analysis of EGF-induced EGFR ubiquitination by mass spectrometry revealed that ubiquitinated lysine residues were located in the TKD of EGFR. However, the specific lysine residues for the CBL-mediated polyubiquitination have not been identified. To investigate the differences in EGFR ubiquitination by ZNRF1 and CBL, we sought to identify the acceptor residues for polyubiquitin chains mediated by these two E3 ubiquitin ligases. We co-transfected HEK293T cells with EGFR and ZNRF1 or CBL, and immunoprecipitated EGFR for liquid chromatography-tandem mass spectrometry analysis.

2021-09-10 | PXD024279 | Pride

Off-the-shelf proximity biotinylation for interaction proteomics.

Project description:Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.

| S-EPMC8373943 | biostudies-literature

β-Actin mRNA interactome mapping by proximity biotinylation.

Project description:The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3' UTR of endogenous MS2-tagged β-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the β-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional β-actin-associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/MARTA2 has been shown to be required for β-actin mRNA localization. We found that FUBP3 binds to the 3' UTR of β-actin mRNA and is essential for β-actin mRNA localization, but does not interact with the characterized β-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

| S-EPMC6600913 | biostudies-literature

Biotinylation by antibody recognition-a method for proximity labeling.

Project description:The high-throughput detection of organelle composition and proteomic mapping of protein environment directly from primary tissue as well as the identification of interactors of insoluble proteins that form higher-order structures have remained challenges in biological research. We report a proximity-based labeling approach that uses an antibody to a target antigen to guide biotin deposition onto adjacent proteins in fixed cells and primary tissues, which allows proteins in close proximity to the target antigen to be captured and identified by mass spectrometry. We demonstrated the specificity and sensitivity of our method by examining the well-studied mitochondrial matrix. We then used the method to profile the dynamic interactome of lamin A/C in multiple cell and tissue types under various treatment conditions. The ability to detect proximal proteins and putative interactors in intact tissues, and to quantify changes caused by different conditions or in the presence of disease mutations, can provide a window into cell biology and disease pathogenesis.

| S-EPMC5790613 | biostudies-literature

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