Principles, Evolution, and Applications of Proximity Labeling
In cell biology, a deep understanding of protein interactions is crucial for revealing complex biological processes within the cell. Traditional methods, such as Co-immunoprecipitation (Co-IP) and pull-down experiments, each have their strengths, but also limitations. For example, Co-IP has high specificity requirements for antibodies and may miss transient or weak interactions, while pull-down experiments can be performed in vitro, but their results may not fully reflect the in vivo situation. As a result, researchers have turned to developing new technologies to more comprehensively explore the protein interaction network (interactome).
Proteins carry out biological functions through complex and dynamic interaction networks, and these interactions often occur instantaneously, making them difficult to capture using traditional methods. Thus, proximity labeling technology (PL) emerged. By achieving rapid and precise labeling in specific subcellular environments, it allows researchers to capture transient protein interactions. This technology not only provides new tools and perspectives for studying protein interactions but also greatly accelerates our understanding of important biological processes like cell signaling and metabolic regulation.
Principles and Classification of Proximity Labeling Technology
The core principle of proximity labeling technology is to use specific enzymes to covalently link labels (such as biotin) to proteins near the target protein, enabling the labeling and subsequent analysis of adjacent proteins. This labeling can be detected by mass spectrometry, revealing interactions between proteins. Based on whether hydrogen peroxide (H₂O₂) stimulation is required, proximity labeling technologies can be divided into two main categories:
Hydrogen Peroxide-Dependent Technologies: Represented by APEX (Ascorbate Peroxidase). APEX is a peroxidase that catalyzes a labeling reaction in the presence of hydrogen peroxide, generating free radicals that covalently react with nearby proteins, thus achieving proximity labeling. APEX technology is fast, but hydrogen peroxide may be toxic to cells, so strict control of conditions is required to avoid oxidative stress.
Hydrogen Peroxide-Independent Technologies: Includes BioID and its derivative techniques (such as BioID2, Split-BioID, etc.) and TurboID and its derivatives (such as miniTurbo, Split-TurboID, etc.). These technologies use biotin ligases to catalyze the chemical reaction between ATP and biotin, linking biotin to the lysine residues of proteins. When these ligases are fused with the target protein, they can transfer biotin to other proteins near the target. These technologies do not require external stimulation, making them easier to operate and more versatile, and have become mainstream.
Evolution of Proximity Labeling Technology
Since its development, proximity labeling technology has gone through multiple iterations, progressively improving labeling efficiency, spatial and temporal resolution, and cellular compatibility. Here are several key technological advancements:
BioID: In 2012, researchers used a mutant form of E. coli biotin ligase, BirA*, to develop the earliest hydrogen peroxide-independent proximity labeling technology, BioID. This technology can label proteins within a 10nm range of the target protein, making it suitable for analyzing steady-state protein interactions. Although the reaction time for BioID is relatively long (usually 18-24 hours), it provides an important tool for studying weak-affinity or transient protein interactions.
BioID2: Developed in 2016, BioID2 significantly reduced the molecular weight of the biotin ligase (from 35kDa to 27kDa), improving its adaptability in small subcellular spaces. While its labeling efficiency is similar to BioID, its smaller size makes it better suited for studies of subcellular structures.
Split-BioID: In 2017, researchers split the BirA enzyme into two parts, which only become active when they bind via protein-protein interactions. This technology allows for precise labeling of specific protein interactions, making it a powerful tool for exploring multiprotein complexes in complex biological systems.
TurboID and miniTurbo: In 2018, researchers selected TurboID and miniTurbo through directed evolution, resulting in significantly higher labeling efficiency. In particular, TurboID can complete labeling in as little as 10 minutes. This high temporal resolution makes it suitable for studying rapidly occurring protein interactions and cellular signaling pathways.
APEX and APEX2: APEX technology uses peroxidase-catalyzed labeling, enabling rapid labeling of nearby proteins in the presence of hydrogen peroxide. The improvement of APEX2 increased its labeling efficiency and stability, allowing it to work at lower hydrogen peroxide concentrations, thus reducing cellular toxicity. APEX and APEX2 are widely used in high temporal and spatial resolution proteomic analysis.
Split-TurboID and Other Emerging Technologies: As research has advanced, various emerging proximity labeling tools, such as Split-TurboID, ProtA-TurboID, BASU, and AirID, have been developed. These new technologies each have unique features, such as Split-TurboID, which splits the TurboID enzyme into two fragments that only re-activate the enzyme's function when an interaction occurs, increasing the specificity of the labeling.