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Cells Apr 2020BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the... (Comparative Study)
Comparative Study
BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.
Topics: A549 Cells; Animals; Biotinylation; Genetic Vectors; Humans; Ligases; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Proteomics
PubMed: 32344865
DOI: 10.3390/cells9051070 -
Nature Communications Feb 2022Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein...
Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.
Topics: Biotinylation; Chromatin; Chromatography, Affinity; Gene Expression Regulation; Gene Regulatory Networks; HEK293 Cells; Humans; Mass Spectrometry; NFI Transcription Factors; Protein Interaction Maps; Proteomics; Transcription Factors
PubMed: 35140242
DOI: 10.1038/s41467-022-28341-5 -
Current Protocols in Protein Science Feb 2018BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to...
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2018 by John Wiley & Sons, Inc.
Topics: Animals; Biotinylation; Carbon-Nitrogen Ligases; Escherichia coli; Escherichia coli Proteins; Humans; Recombinant Fusion Proteins; Repressor Proteins
PubMed: 29516480
DOI: 10.1002/cpps.51 -
Cell Metabolism Sep 2020We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human...
We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles.
Topics: Biotinylation; Cells, Cultured; HEK293 Cells; Humans; Mitochondria; Mitochondrial Proteins; Protein Interaction Maps
PubMed: 32877691
DOI: 10.1016/j.cmet.2020.07.017 -
Science (New York, N.Y.) Mar 2013Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant...
Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.
Topics: Animals; Ascorbate Peroxidases; Biotinylation; COS Cells; Chlorocebus aethiops; Gene Targeting; Genetic Engineering; HEK293 Cells; Humans; Mass Spectrometry; Mitochondria; Proteomics
PubMed: 23371551
DOI: 10.1126/science.1230593 -
Methods in Molecular Biology (Clifton,... 2019BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin...
BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin ligase, called BioID, fused to a protein of interest that when expressed in cells can be induced to biotinylate interacting and proximate proteins over a period of hours, thus generating a history of protein associations. These biotinylated proteins are subsequently purified and identified via mass spectrometry. Compared to other conventional methods typically used to screen strong PPIs, BioID allows for the detection of weak and transient interactions within a relevant biological setting over a defined period of time. Here we briefly review the scientific progress enabled by the BioID technology, detail an updated protocol for applying the method to proteins in living cells, and offer insights for troubleshooting commonly encountered setbacks.
Topics: Animals; Biotinylation; Cell Line, Tumor; Humans; Mice; Protein Binding; Protein Interaction Mapping; Proteins; Staining and Labeling
PubMed: 31161514
DOI: 10.1007/978-1-4939-9546-2_15 -
Cell Dec 2015The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular...
The centrosome is the primary microtubule organizing center of the cells and templates the formation of cilia, thereby operating at a nexus of critical cellular functions. Here, we use proximity-dependent biotinylation (BioID) to map the centrosome-cilium interface; with 58 bait proteins we generate a protein topology network comprising >7,000 interactions. Analysis of interaction profiles coupled with high resolution phenotypic profiling implicates a number of protein modules in centriole duplication, ciliogenesis, and centriolar satellite biogenesis and highlights extensive interplay between these processes. By monitoring dynamic changes in the centrosome-cilium protein interaction landscape during ciliogenesis, we also identify satellite proteins that support cilia formation. Systematic profiling of proximity interactions combined with functional analysis thus provides a rich resource for better understanding human centrosome and cilia biology. Similar strategies may be applied to other complex biological structures or pathways.
Topics: Biotinylation; Cell Cycle; Centrosome; Cilia; Humans; Microtubule-Organizing Center; Protein Interaction Maps
PubMed: 26638075
DOI: 10.1016/j.cell.2015.10.065 -
Nature Communications May 2022Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We...
Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.
Topics: Animals; Astrocytes; Biotin; Biotinylation; Brain; Mice; Neurons; Proteome; Proteomics
PubMed: 35614064
DOI: 10.1038/s41467-022-30623-x -
Journal of Proteomics May 2023Proximity biotinylation screens are a widely used strategy for the unbiased identification of interacting or vicinal proteins. The latest generation biotin ligase...
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 immobilized 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. SIGNIFICANCE: Proximity biotinylation screens using the last generation biotin ligase TurboID represent a powerful approach for the characterisation of transient protein-protein interaction and signaling networks. However, a constant and high basal biotinylation rate and the associated cytotoxicity often preclude the use of this method in proteomic studies. We report a protocol based on modulation of free biotin levels that prevents the deleterious effects of TurboID while allowing inducible biotinylation, even in subcellular compartments such as the endoplasmic reticulum. This optimised protocol greatly expands the applications of TurboID in proteomic screens.
Topics: Humans; Biotinylation; Biotin; Proteomics; Proteins; Ligases
PubMed: 36966971
DOI: 10.1016/j.jprot.2023.104886 -
Biochemical and Biophysical Research... Dec 2017Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to...
Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.
Topics: Animals; Biotin; Biotinylation; Blotting, Western; Carbon-Nitrogen Ligases; Dogs; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Hot Temperature; Immunoprecipitation; Madin Darby Canine Kidney Cells; Proteins; Recombinant Proteins; Repressor Proteins; Solubility; Streptavidin
PubMed: 28986262
DOI: 10.1016/j.bbrc.2017.09.168