Profiling palmitoyltransferase ZDHHC4-interacting proteins by TurboID-mediated proximity labeling strategy
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Aimin Yang
PROVIDER: PXD056770 | iProX | Sat Oct 12 00:00:00 BST 2024
REPOSITORIES: iProX
Similar Datasets
Project description:RRS1 was fused with highly active biotin ligase TurboID tagged with a FLAG epitope for proximity labeling.Biotin treatment of transgenic seedlings induced the overall biotinylation output signal.The total biotinylated proteins were enriched using streptavidin-conjugated beads and analyzed via liquid chromatography/tandem mass spectrometry analysis.
2025-01-03 | MSV000096770 | MassIVE
Project description:HDMB03 cells were transiently transfected with plasmids expressing NLS-TurboID or 3xHA-TurboID-OTX2 under the control of a strong CAG promoter. After a 15-minute pulse with 500 µM exogenous biotin, RIPA lysates were prepared and processed with streptavin-sepharose to capture biotinylated proteins that were identified by using mass spectrometry.
2024-05-31 | PXD052687 | Pride
Project description:This project presents a comprehensive profiling of pre-miRNA156a-interacting proteins using an integrated MCP-MS2 and TurboID proximity labeling approach in vitro. We performed fusion expression of pre-miRNA with MS2, and co-expressed MCP-TurboID. Due to the specific binding between MCP and MS2, TurboID is brought into close proximity with pre-miRNA. Upon exogenous addition of biotin, interacting molecules of pre-miRNA are biotinylated by TurboID. After cell lysis and incubation with streptavidin magnetic beads, the biotinylated proteins are enriched. Finally, mass spectrometry (MS) analysis is performed to identify the molecules interacting with pre-miRNA, thereby capturing the interaction profile of pre-miRNA-associated proteins.
2025-07-14 | PXD066118 |
Project description:Proximity biotinylation is a commonly used method to identify the in vivo proximal proteome for proteins of interest. This technology typically relies on fusing a bait protein to a biotin ligase using overexpression or CRISPR-based tagging, thus prohibiting such assays in (primary) cell types that are difficult to transfect. We recently developed an ‘off the shelf’ proximity biotinylation method, which makes use of a recombinant enzyme consisting of the biotin ligase TurboID fused to the antibody-recognizing moiety Protein A. In this method, a bait specific antibody and the ProteinA-Turbo enzyme are consecutively added to permeabilized fixed or unfixed cells. Following incubation, during which ProteinA-Turbo-antibody-antigen complexes are formed, unbound molecules are washed away, after which bait-proximal biotinylation is triggered by the addition of exogenous biotin. Finally, biotinylated proteins are enriched from crude lysates using streptavidin beads followed by mass spectrometry-based protein identification. Here, we present a detailed protocol for this method
2022-10-17 | PXD030963 | Pride
Project description:To review an improved proximity-labeling strategy that uses the improved E. coli biotin ligase TurboID to characterize C. elegans protein complexes, the interactome of a presynaptic active zone protein, ELKS-1, was analyzed as proof of principle. A significant constraint on the sensitivity of TurboID-based proximity labeling is the presence of abundant, endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a co-factor. We developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry, by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography (IMAC).
2022-09-12 | PXD034639 | Pride
Project description:Spleens from the B6 mice were isolated and single cell suspension was made. CD4 T cells were purified from the splenocytes using magnetic bead separation. Briefly, Splenocytes were incubated with biotinylated antibody cocktail consisting of antibodies (Biolegend) to CD19, B220, CD11b, CD11c, NK1.1, Gr1, CD25 CD8. After a wash step, cells were incubated with streptavidin conjugated magnetic particles (BD Biosciences). After washing, CD4 T cells were isolated by applying a magnetic field and removing the untouched cells. Purified CD4 T cells were then activated with plate-bound anti-CD3 plus anti-CD28 in presence of either Th1 or Th17 or Th1/17 polarizing condition for 3 days. Total RNA from the 3 days differentiated Th1, Th17 and Th1/17 cells was isolated using mirVana miRNA isolation Kit (Invitrogen).
2017-09-13 | E-MTAB-5237 | biostudies-arrayexpress
Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 and the nuclear transport factor MEX67 indicated (PMID: 36410438; PXD031245) that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. Here we extended this approach to target NUPs NUP110, NUP76 and NUP96 with a TurboID-HA tandem tag fused to both, the N and C-terminus of each respective target protein. While the central NUP96 biotinylated most nuclear pore proteins with the marked exception of the nuclear basket, NUP158, NUP110 and NUP76 caused biotinylation of specific sub-complexes of the pore and Mex67 labelled NUPs lining the pore channel.
2023-12-04 | PXD047268 | Pride
Project description:Nicotiana benthamiana plants were infiltrated with three different constructs: 1) N-TurboID-3HA 2) NTIR-TurboID-3HA or 3) Citrine-TurboID-3HA. N-TurboID is the full length N-gene fused to TurboID, NTIR-Turbo is the TIR domain of the N-gene fused to turboID, and Citrine-TurboID is the control construct. Three biological replicates for each construct were labeled with TMT10Plex reagents (Lot TB260979). N-TurboID (TMT: 126, 127N, 126C); NTIR-TurboID (TMT: 128N, 128C, 129N); citrine-TurboID (TMT: 129C, 130N, 130C). Biotinolayted proteins were enriched with streptavidin beads (equal protein input onto beads) and digested into peptides with Trypsin/LysC. An equal volume of peptides were labeled with TMT reagents.
2018-10-10 | MSV000083018 | MassIVE
Project description:Nicotiana benthamiana plants were infiltrated with three different constructs: 1) N-TurboID-3HA 2) NTIR-TurboID-3HA or 3) Citrine-TurboID-3HA. N-TurboID is the full length N-gene fused to TurboID, NTIR-Turbo is the TIR domain of the N-gene fused to turboID, and Citrine-TurboID is the control construct. Three biological replicates for each construct were labeled with TMT10Plex reagents (Lot TB260979). N-TurboID (TMT: 126, 127N, 126C); NTIR-TurboID (TMT: 128N, 128C, 129N); citrine-TurboID (TMT: 129C, 130N, 130C). Biotinolayted proteins were enriched with streptavidin beads (equal protein input onto beads) and digested into peptides with Trypsin/LysC. An equal volume of peptides were labeled with TMT reagents.
2018-10-10 | MSV000083019 | MassIVE
Project description:To characterize the nascent transcriptome during zygotic genome activation (ZGA) of Xenopus laevis embryos, we microinjected 5-ethynyl uridine (EU) into 1-cell stage embryos and isolated total RNAs from whole embryos at 5, 6, 7, 8 and 9 hours post-fertilization (hpf) at room temperature, respectively, covering the stages of pre-ZGA to widespread ZGA (stage 7-9). To purify nascent transcripts, total RNAs were biotinylated using disulfide biotin azide via click reaction and biotinylated nascent transcripts were purified using streptavidin beads. Libraries were constructed from using the total RNA ('All'), nascent transcripts ('Bead') and the flowthrough after purification of nascent transcripts ('FL'), respectively. To categorize maternal-zygotic (MZ) genes and zygotic-only (Z) genes, total RNAs from eggs were isolated for constructing libraries. All libraries were sequenced on illumina NextSeq 500.
2022-08-22 | GSE201835 | GEO