Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Control of TurboID-dependent biotinylation intensity in proximity ligation screens

ABSTRACT: Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase TurboID has broadened the range of potential applications, as this ligase promotes an intense and faster biotinylation, even in subcellular compartments like the endoplasmic reticulum. On the other hand, the uncontrollable high basal biotinylation rates deny the system´s inducibility and are often associated with cellular toxicity precluding its use in proteomics. We report here an improved method for TurboID-dependent biotinylation reactions based on the tight control of free biotin levels. Blockage of free biotin with a commercial biotin scavenger reversed the high basal biotinylation and toxicity of TurboID, as shown by pulse-chase experiments. Accordingly, the biotin-blockage protocol restored the biological activity of a bait protein fused to TurboID in the endoplasmic reticulum and rendered the biotinylation reaction inducible by exogenous biotin. Importantly, the biotin-blockage protocol was more effective than biotin removal with immobilised avidin and did not affect the cellular viability of human monocytes over several days. The method presented should be useful to researchers interested in exploiting the full potential of biotinylation screens with TurboID and other high-activity ligases for challenging proteomics questions.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Thomas Krüger

LAB HEAD: Axel A. Brakhage

PROVIDER: PXD040302 | Pride | 2023-03-29

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

MicroID: A Novel Biotin Ligase Enables Rapid Proximity Ligation Proteomics

Project description:Identifying protein-protein interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity ligation method that uses a promiscuous biotin ligase to detect protein-protein interactions in cells in a highly reproducible manner. Recent advancements in proximity ligation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2 to create a smaller, highly active, biotin ligase that we named MicroID. Truncation of the c-terminal of BioID2 and mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller, highly active construct with lower baseline labeling. Several additional point mutations improved the function of our modified MicroID construct compared to BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID is the smallest biotin ligase (180 AA for microID vs. 257 AA for miniTurbo and 338 AA for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurboID. MicroID also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we developed a MicroID mutant that has lower labeling efficiency, but significantly reduced biotin scavenging compared to BioID2. Finally, we demonstrate utility of MicroID in mass spectrometry experiments by localizing MicroID constructs to subcellular organelles and measuring protein-protein interactions.

2022-08-12 | PXD031146 | Pride

Thiol-cleavable Biotin for Chemical and Enzymatic Biotinylation and its Application to Mitochondrial TurboID Proteomics

Project description:Protein biotinylation via chemical or enzymatic reactions is often coupled with streptavidin-based enrichment and on-beads digestion in numerous biological applications. However, the popular on-beads digestion method faces major challenges of streptavidin contamination, the lost information of biotinylation sites, and limited sequence coverage of enriched proteins. Here, we explored thiol-cleavable biotin as an alternative approach to elute biotinylated proteins from streptavidin-coated beads for both chemical biotinylation and biotin ligase-based proximity labeling. All possible amino acid sites for biotinylation were thoroughly evaluated besides the primary lysine residue. We found that biotinylation at lysine residues notably reduces the trypsin digestion efficiency which can be mitigated by the thiol-cleavable biotinylation method. We then evaluated the applicability of thiol-cleavable biotin as a substrate for proximity labeling in living cells, where TurboID biotin ligase was engineered onto the mitochondrial inner membrane facing the mitochondrial matrix. As a proof-of-principle study, thiol-cleavable biotin-assisted TurboID proteomics achieved remarkable intra-organelle spatial resolution with significantly enriched proteins localized in the mitochondrial inner membrane and mitochondrial matrix.

2021-04-21 | MSV000087256 | MassIVE

Identification of myelin basic protein interactome using TurboID proximity labeling proteomics

Project description:The protein environment of myelin basic protein (MBP), its uncharged form, and cyclindependent kinase inhibitor 1 (p21Cip1) were analyzed using two different approaches to identify protein partners thereof. The first one was a state-of-the-art enzymatic approach (TurboID), which utilizes an engineered biotin ligase that uses ATP to convert biotin to reactive biotin-AMP that covalently attaches to nearby proteins, followed by enrichment of biotin-labeled proteins on streptavidin beads and MS analysis of the resulting protein pool. Classical immunoprecipitation with Flag-tag fused to the target protein in combination with MS analysis was used as a reference method. As a result, two pools of potential interactors of MBP and uncharged MBP were identified. It was shown that TurboID, although giving a high level of background biotinylation, is a more reproducible technique compared to the immunoprecipitation, possibly due to higher affinity during the fraction enrichment process and easier maintaining of uniform conditions for the process, as well as interaction conditions being closer to in vivo conditions.

2023-01-25 | PXD039467 | Pride

Off the shelf proximity biotinylation using ProtA-TurboID

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

Identification of biotinylated proteins through pull-down: ID and label-free quantification (Identification of TTP-interactome in RAW264.7 cells by TurboID proximity labelling).

Project description:Identification of biotinylated proteins through pull-down. RAW264.7 cells stably expressing a Dox-inducible TurboID-TTP fusion protein were treated with LPS or Epoxomicin for 4 h, followed by 15 min of biotin labeling in the cell medium. TurboID without fusion was used as a control.

2023-03-24 | PXD036714 | 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 of interest using an endogenous antibody against the bait protein. 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, the histone modification H3K9me3 and the chromatin remodeler BRG1. Amongst the main findings, our experiments revealed Flywch1 as an essential protein that interacts with H3K9me3-marked centromeric heterochromatin. 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.

2021-07-12 | PXD025012 | Pride

Identification of interaction partners of the Toxoplasma gondii Cysteine Repeat Modular Proteins TGGT1_292020 and TGGT1_261080

Project description:Apicomplexa are obligate intracellular parasites. While most species are restricted to specific hosts and cell types, Toxoplasma gondii can invade every nucleated cell derived from warm-blooded animals. This broad host range suggests that this parasite can recognize multiple host cell ligands or structures, leading to the activation of a central protein complex, which should be conserved in all apicomplexans. Cysteine Repeat Modular Proteins (CRMPs) are a family of apicomplexan specific proteins. In Toxoplasma gondii, two CRMPs are present in the genome. We performed pulldown of 3xHA tagged CRMPA and CRMPB. In a second dataset we performed biotinylation of TurboID tagged CRMPB on extracellular parasites to identify transient interaction partners. In a third dataset, we performed biotinylation of TurboID tagged CRMPA or B during invasion or in an invasion blocking buffer.

2022-12-13 | PXD031649 | Pride

Off-the-shelf proximity labeling

Project description:In this study we developed a new assay to perform proximity biotinylation in cells without the requirement for genetic manipulation or transfection to fuse a biotin ligase to a protein of interest. We show the specificity by targeting H3K9me3 and performing ChIP-seq for the biotin modification. Targeting IgG was used as a control. By doing so, we identified flywch1 as a new protein binding to H3K9me3 regions, which we validated using ChIP-seq (IgG ChIP was used as a control)

2021-07-09 | GSE169317 | GEO

Proximity-dependent biotinylation by TurboID to identify protein-protein interaction networks in yeast

Project description:The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interactions (PPI) studies. Yet, despite the recurrent and successful use of BioID-based PPI screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze PPIs in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

2019-05-16 | PXD013183 | Pride

Establishment of TurboID proximity proteomics in the filamentous fungus Sordaria macrospora

Project description:Biotin identification (BioID) is a proximity-dependent labeling technique to study protein-protein co-localization in vivo. Although BioID has been applied in animal cells, plants and yeast, the method remained to be established in filamentous fungi. In this study, we established BioID for the filamentous fungus Sordaria macrospora using the well-characterized striatin interacting phosphatase and kinase (STRIPAK) complex as a proof of principle. In detail, the STRIPAK complex interactor 1 (SCI1) was fused to a codon-optimized TurboID biotin ligase. This SCI1-TurboID fusion protein complemented the Δsci1 deletion strain phenotype. The identification of the already known SmSTRIPAK components PRO11, SmMOB3, SmPP2Ac1 and PRO22 in a BioID experiment with SCI1-TurboID demonstrated its successful application in S. macrospora. The technique will provide a powerful proteomics tool for fungal biologists.

2022-10-25 | PXD034217 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data