BioID-MS Service

What Is BioID-MS

Building a comprehensive protein interaction map is crucial for understanding cellular function. Challenges include considering direct binary protein-protein interactions and indirect interactions (e.g., within the same multi-protein complex) and neighboring protein networks. Discovery methods must account for dynamic properties in cellular processes and overcome technical challenges posed by weak protein-protein interactions. BioID-MS(Proximity Protein Labeling-mass spectrometry), a proximity-dependent biotin labeling technology, addresses these issues.

Core advantages of BioID-MS include minimal disruption to cell viability during PPI detection in the native cellular environment. Adjustable detection range by modifying fusion protein linker lengths adds versatility. This technology holds significant significance in proteomics research.

BioID-MS excels in identifying weak, transient, insoluble, and post-translation modified protein interactions, potentially surpassing AP-MS in detecting low-abundance proteins. It has been applied in coronavirus replication, HIV protein interaction networks, bacterial host recognition, and mitosis-related protein interactions. However, low-expressing PPI partners may lead to false negatives, and biotinylation itself may impact protein behavior/interactions in certain cases.

The Principle of BioID-MS

The principle of BioID-MS involves the fusion of a target protein with a bacterial biotin ligase (BirA). Upon expression of the fusion protein in cells, proteins in proximity to the target protein are biotinylated and labeled with biotin. Subsequently, the biotinylated proteins are captured using streptavidin-coated solid supports, allowing the separation of biotinylated proteins. Finally, mass spectrometry is employed to identify the interacting proteins of the target protein.

(Ummethum H, Hamperl S. Proximity Labeling Techniques to Study Chromatin. Front Genet. 2020 May 12;11:450.)

Technical Workflow Of BioID-MS

(1) Construct an expression vector for the BioID fusion protein, stably expressing the target protein fused with BirA.

(2) Induce biotinylation of proteins near the target protein by adding biotin under specific conditions.

(3) Extract total protein and incubate it with streptavidin-coated magnetic beads to enrich biotinylated proteins.

(4) Identify the enriched biotinylated proteins using Western blot and mass spectrometry analysis methods.

(Mujeeb R. Cheerathodi et al,. BioID Combined with Mass Spectrometry to Study Herpesvirus Protein–Protein Interaction Networks Methods in molecular biology 2020)

Advantages of BioID-MS Service

Capturing protein species or large molecular complexes interacting with bait proteins in a more physiological state.

Utilizing biotin-streptavidin non-covalent binding, allowing the use of strong detergents to wash samples and improve signal-to-noise ratio.

Even with transient interactions undergoing displacement, biotinylation characteristics are retained, enabling BioID-MS to achieve a transient interaction landscape, facilitating higher temporal and spatial resolution for in vivo applications.

Applications of BioID-MS

Studying protein-protein interactions.

Constructing protein interaction networks.

Screening for weak or transient interactions (e.g., identifying kinase substrates).

Revealing mechanisms of pathogen invasion, replication, and immune evasion.

Cell screening in various animal cells.

Delivery

Project reports

Protein elution liquid silver-stained gel images

Mass spectrometry raw data and analysis results

Protein-protein interaction (PPI) network diagrams

Comparison of BioID Technology with Other Protein Interaction Studies

Traditional protein interaction research techniques, such as Yeast-Two-Hybrid, lack a post-translational modification system similar to mammalian cells and rely on high-quality cDNA libraries. Immunoprecipitation-Mass Spectrometry (IP-MS) is limited by antibody quality and struggles to preserve spatial localization information of protein interactions and capture weaker or transient interactions.

Compared to traditional interaction discovery methods, BioID exhibits significant advantages, especially in identifying transient or weak interactions, and is suitable for insoluble subcellular structures, significantly impacting our understanding of cell structure and function.

Despite being proximity-dependent, biotinylation by BioID does not require direct or indirect interaction between the BioID fusion protein and the protein to be biotinylated. Even in the absence of direct interactions, neighboring proteins are labeled, providing useful information about the protein landscape surrounding the target protein.

Proteins with transient interactions or transient proximity to the BioID fusion protein will also be biotinylated. Importantly, biotinylation persists even after the interaction has ceased or the protein has left the vicinity of the BioID fusion protein. Thus, BioID is well-suited for studying dynamic cellular processes in both spatial and temporal aspects.

Unlike more traditional methods such as affinity purification, strict extraction conditions in those methods can disrupt protein-protein interactions and lead to data loss. However, BioID allows for rigorous conditions during cell lysis and subsequent streptavidin binding to separate biotinylated proteins. Therefore, BioID has been particularly useful for studying insoluble protein structures, such as nucleoli or centrosomes.

References

1. In Situ Capture of Chromatin Interactions by Biotinylated dCas9;
2. Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity;
3. Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation;
4. Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling;
5. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells
6. AirID, a novel proximity biotinylation enzyme, for analysis of protein–protein interactions

In Situ Capture of Chromatin Interactions by Biotinylated dCas9

Liu et al. fused AVI with dCas9 and combined it with BirA expression to target specific genomic loci with designed sgRNA. They utilized high-affinity streptavidin purification to isolate macromolecular complexes associated with the genomic sites of interest. By employing mass spectrometry (MS)-based proteomics and high-throughput sequencing, they identified and analyzed the purified protein, RNA, and DNA complexes, enabling the study of natural cis-regulatory protein, RNA, and long-range DNA interactions.

The authors utilized validated telomere-targeting sgRNA (sgTelomere; Figure 1C), demonstrating specific labeling of telomeres by the dCas9-EGFP fusion protein in contrast to diffuse nucleolar localization of non-targeted dCas9-EGFP (Figure 1D). After co-expression of sgTelomere and biotinylated dCas9, significant enrichment of telomere DNA was observed (Figure 1E). The known telomere-associated protein TERF2 was highly enriched in samples expressing sgTelomere but absent in control samples (Figure 1F). Importantly, using iTRAQ-based proteomics, several known telomere maintenance proteins and novel telomere-associated proteins were identified.

BirA has been newly applied to label specific protein groups: targeting chromatin-associated proteins by directing BirA to specific AviTag-labeled nucleosomes, enabling biotinylation (1); targeting a synaptic membrane to visualize specific protein-protein interactions at synapses by biotinylating AviTag proteins on the opposing synaptic membrane (2); expressing BirA substrate peptides and BirA on nuclear membrane proteins in specific tissues of Arabidopsis, C. elegans, or Drosophila, enabling pull-down of nuclei from specific cell types (3); furthermore, using enzymes for promiscuous biotinylation (BirA mutants or peroxidases) has allowed labeling of untagged proteins in specific cellular regions or compartments (4).