Principles, Evolution, and Applications of Proximity Labeling
Specific Applications of Proximity Labeling Technology
Protein Interaction Network Research: Proximity labeling technology can capture transient or low-affinity protein interactions, overcoming the limitations of traditional techniques. For example, researchers can use TurboID technology to quickly screen for molecules that interact with a specific protein, and identify these interacting proteins through mass spectrometry, helping to uncover key nodes in signaling pathways or metabolic regulatory networks.
Subcellular Proteomics: Technologies such as BioID and APEX are widely used to study the composition of proteins in subcellular organelles. For instance, researchers can direct APEX to mitochondria, the endoplasmic reticulum, or nuclear pore complexes, and identify specific proteins in these subcellular structures through labeling and mass spectrometry, revealing the roles these structures play in cellular physiological functions.
Spatiotemporal Study of Dynamic Protein Interactions: Using techniques like TurboID and APEX2, researchers can capture dynamic protein interactions that occur within a short time frame. This is crucial for studying processes such as signal transduction and cell cycle regulation. For example, in the insulin signaling pathway, researchers used TurboID to label proteins interacting with the insulin receptor, uncovering key regulatory factors in signal transmission.
Photocatalytic Proximity Labeling Technology: Light-sensitive proximity labeling techniques use light to trigger labeling reactions, offering good spatial specificity. These methods are particularly suitable for studying dynamic protein-RNA and protein-DNA interactions within cells, showing great potential in research areas like gene expression regulation.
Examples of Applications
In 2023, Yanting Su et al. published an article titled “Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology” in BMC Genomics (PubMed ID: 36964488). The study utilized TurboID-based proximity labeling technology to screen and validate proteins interacting with FOXO1, providing important information for further understanding the function of FOXO1 and the regulatory networks it participates in.
In 2022, Fujian Lu et al. published an article titled “CMYA5 establishes cardiac dyad architecture and positioning” in Nature Communications (PubMed ID: 35449169). The study applied BioID-based proximity labeling technology in mouse hearts and used proteomics to identify proteins enriched in specific structures, revealing that CMYA5 is a protein closely associated with RYR2, essential for normal heart function.
In 2023, Sammy El-Mansi et al. published an article titled “Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor” in Blood (PubMed ID: 36564030). The study combined APEX2 proximity labeling with innovative dual functional knockout screening, successfully identifying proteins associated with actomyosin ring function, providing new insights into the mechanism of actomyosin-mediated expulsion of von Willebrand factor.
In 2023, Tongyu Sun et al. published an article titled “Crosstalk between RNA m6A and DNA methylation regulates transposable element chromatin activation and cell fate in human pluripotent stem cells” in Nature Genetics (PubMed ID: 37474847). This study combined multiple CRISPR targeting methods, Chimeric Array of gRNA Oligos (CARGO), with biotin proximity labeling (BioID) technology to develop a CRISPR-based, transposable element-centered proteomics method called CARGO-BioID. They designed the CARGO construct to express up to 15 sgRNAs to target transposable element (TE) sequences in native chromatin environments and fused catalytically inactive dead Cas9 (dCas9) with biotin ligase TurboID, expecting the fusion protein to biotinylate proteins near both the target and the DNA sequences it directs. By streptavidin affinity purification, these biotinylated proteins were subjected to LC-MS/MS analysis to identify proteins specifically enriched at targeted TE sites. After three hours of exogenous biotin treatment, the researchers observed differences in biotinylation patterns of nuclear proteins in human embryonic stem cells (hESCs) expressing dCas9-TurboID-CARGO sgRNAs compared to those with control sgRNAs, and identified 144 candidate proteins highly enriched at the LTR7 site. These proteins were involved in histone modification, chromatin organization, transcription initiation, mRNA processing, and pluripotency factor signaling. To avoid disrupting endogenous interactomes (such as transcription factors) potentially affected by dCas9-TurboID binding to DNA, they designed sgRNAs to avoid predicted transcription factor binding motifs, confirming that CARGO-BioID did not interfere with transcription and epigenetic activities. The plasmid sequences used in this paper, such as Lenti-dCas9-TurboID-blast, are available at Addgene 440259.