How Can TurboID-Based Proximity Labeling Enable the Study of Low-Abundance Protein Interactions?
- expression of a bait-TurboID fusion protein
- supplementation of excess biotin
- TurboID-mediated covalent transfer of activated biotin (bio-AMP) to lysine residues within a radius of ~10 nm surrounding the bait
- enrichment of biotinylated proximal proteins using streptavidin magnetic beads followed by mass spectrometry analysis
- Customized TurboID vector construction and stable cell line generation
- Optimized biotin labeling protocols
- High-specificity streptavidin-based enrichment and protein purification
- High-resolution Orbitrap mass spectrometry (supporting DIA/SPS-MS3)
- Network visualization and functional annotation analyses
Low-abundance proteins constitute essential regulators within the cellular protein network, including transcription factors, signaling modulators, and epigenetic regulatory proteins. However, their intrinsically low abundance, spatiotemporally restricted expression, and transient or weak interactions with binding partners make conventional affinity-based approaches such as immunoprecipitation (Co-IP) and pull-down insufficient to recover their genuine interaction networks. The development of proximity labeling (PL) technologies, particularly the introduction of TurboID, provides an effective solution to this challenge. TurboID does not require direct or stable protein-protein binding; instead, it enables the in situ labeling of proteins within the local neighborhood of a bait protein in living cells, thereby supporting interaction mapping with high spatial and temporal resolution and enhancing the feasibility of studying low-abundance protein interactions.
What Is TurboID?
TurboID is an engineered biotin ligase optimized from BioID. Whereas BioID typically requires prolonged incubation (18-24 hours) to complete biotinylation, TurboID achieves efficient labeling within approximately 10 minutes at physiological temperature, substantially improving temporal resolution and minimizing background labeling.
The general workflow involves:
This strategy represents a shift from binding-dependent to proximity-dependent interaction profiling and is particularly advantageous for capturing dynamic, transient, weak, or membrane-associated protein complexes that are difficult to resolve using traditional affinity-based approaches.
How Does TurboID Facilitate the Study of Low-Abundance Protein Interactions?
1. Sensitive Detection of Weak and Transient Interactions
TurboID labeling requires only spatial proximity rather than stable complex formation, enabling the detection of transient interactions between low-abundance regulatory factors, such as transcription factors and signaling proteins, and their targets.
2. High Spatial and Temporal Resolution
By employing cell-type-specific promoters, subcellular targeting sequences, or inducible expression systems, TurboID enables interaction profiling at defined spatial compartments and discrete time points.
3. Enhanced Enrichment Efficiency for Low-Abundance Protein Interactions
TurboID-labeled proteins can be purified through the ultra-high affinity of streptavidin toward biotin, markedly improving the recovery of low-abundance interactors. Compared with Co-IP, which depends heavily on antibody performance and protein abundance, TurboID exhibits greater applicability and enrichment efficiency.
4. Compatibility with Advanced Mass Spectrometry Platforms
Coupling TurboID with high-sensitivity Orbitrap platforms (e.g., Orbitrap Exploris 480) and data-independent acquisition (DIA) workflows enables deeper coverage, higher reproducibility, and quantitative identification of low-abundance biotinylated proteins.
How Can Common TurboID-Related Pitfalls Be Avoided?
Despite its high labeling efficiency, the elevated catalytic activity of TurboID can introduce non-specific labeling and high background. Key recommendations include:
1. Rigorous control design, including empty TurboID vector, catalytically inactive mutant, or biotin-withdrawn controls to remove background signals.
2. Controlled expression levels to avoid aberrant localization or aggregation due to overexpression.
3. Optimization of biotin concentration and incubation duration, as 10-15 minutes at 50-500 µM typically suffices and helps minimize non-specific labeling.
4. Optimization of enrichment and mass spectrometry processing, including the use of high-affinity streptavidin magnetic beads and mild elution conditions to reduce non-specific contaminants.
TurboID Platforms at MtoZ Biolabs for Low-Abundance Protein Interactions Analysis
At MtoZ Biolabs, we have established an integrated TurboID workflow encompassing:
We have developed enrichment strategies and data-processing pipelines specifically tailored for low-abundance interaction profiling to enable reliable signal extraction from background and to reveal previously inaccessible layers of interactome regulation.
TurboID-based proximity labeling is rapidly emerging as a gold-standard approach for low-abundance protein interactions. Its high spatial and temporal precision, enhanced enrichment efficiency, and robust compatibility with mass spectrometry offer new opportunities for dissecting cellular signaling pathways and disease mechanisms. MtoZ Biolabs is committed to providing researchers with one-stop solutions spanning vector construction, mass spectrometry characterization, and computational analysis to accelerate the discovery of critical interaction targets and facilitate scientific innovation.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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