Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

Kim, Dae In; Roux, Kyle J.

doi:10.1016/j.tcb.2016.09.004

Cited by 248 publications

(231 citation statements)

References 94 publications

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“…Proximity labeling provides a means to capture the immediate biochemical environment of a protein as it exists in situ, thus preserving the critical spatial and temporal context (Kim and Roux, 2016). Various methods have been developed but, among them, engineered ascorbic acid peroxidase (APEX) is of particular interest because of its rapid labeling kinetics (Lam et al, 2015; Martell et al, 2012; Rhee et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

Lobingier

Hüttenhain

Eichel

et al. 2017

Cell

357

334

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SUMMARY Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

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Section: Introductionmentioning

confidence: 99%

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

Lobingier

Hüttenhain

Eichel

et al. 2017

Cell

357

334

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“…Enzyme-mediated ligation methods can be used for several distinct purposes including building peptide/protein assemblies (1,2), making site-specific protein modifications (3,4), and elucidating protein-protein interactions (PPIs) (see (5) for review). Proteins regularly form complex and specific networks critical to the structural and functional integrity of the cell.…”

Section: Introductionmentioning

confidence: 99%

“…Since its development and initial application in 2012 (7), BioID has been cited and/or applied in over 200 PubMed-published articles and has been utilized in several unicellular organisms (see (12–15) for examples), mammalian cells (5), plants (16,17), and mice (18–20), including compartmental proteomics of a parasitic organism infecting mice (21). Novel or improved applications using BioID ligase have spurred numerous follow-up articles including a smaller version of BioID with improved sensitivity and localization (22), its use for identifying protein-RNA interactions (23), split-BioID studies (24,25), and faster versions of BioID (26).…”

Section: Introductionmentioning

confidence: 99%

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BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

Self Cite

107

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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.

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“…1A). This proximal biotinylation has been successfully used to identify novel protein interactions [see reviews: 4, 9], and has been shown as complementary to traditional affinity purification approaches [10]. Interests in this approach have led to improved variants of BioID proteins: BioID2, a smaller biotin ligase from Aquifex aeolicus [11], and a split-BioID as a dimerization dependent biotin ligase [12, 13].…”

Section: Introductionmentioning

confidence: 99%

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat

Cheah

Yamada

2017

Biochemical and Biophysical Research Communications

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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.

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Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

Cited by 248 publications

References 94 publications

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells

BioID as a Tool for Protein-Proximity Labeling in Living Cells

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat

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