Proximity Labeling (PL) is an emerging technique for screening protein-protein interactions (PPI).

Keywords: Proximity Labeling Technology

Technical Overview

Proximity Labeling (PL) is an emerging technique for screening protein-protein interactions (PPI). The principle includes fusing a proximity-labeling enzyme (biotin ligase) with a target protein. Through enzyme-catalyzed covalent modification, proteins in close proximity to the bait protein are labeled with biotin. Subsequently, biotin-labeled proteins are enriched using streptavidin magnetic beads and identified via mass spectrometry. Biotin is a natural coenzyme that binds tightly to glycoproteins, streptavidin, or similar proteins, facilitating the purification and identification of biotin-labeled proteins.

Applications

- Investigating interaction partners of target proteins

- Studying interactions of disease-critical or drug-target proteins

- Discovering novel biomarkers or target proteins

- Researching tissue-specific secreted proteins

Experimental Workflow

(1) Vector Construction

Construct a plasmid expressing the target protein fused with HA-tagged TurboID. The HA tag helps to verify the expression of the fusion protein in cells.

(2) Stable Cell Line Generation

Introduce the plasmid into a suitable cell line to achieve stable expression of the fusion protein. Confirm expression via the HA tag. The chosen cell line should be appropriate for the research focus and amenable to transfection. Experimental groups include a treatment group (expressing the fusion protein) and a control group (expressing a control plasmid lacking the target gene) to eliminate proteins that bind to TurboID itself.

(3) Biotinylation Treatment

Under appropriate catalytic conditions, proteins in proximity to the target protein are labeled with biotin. These biotinylated proteins are then enriched using streptavidin magnetic beads.

(4) Proteomic Analysis

Each group includes three biological replicates, totaling six samples for proteomic analysis to identify proteins interacting with the target protein.

Advantages

- Alternative to co-immunoprecipitation (co-IP); does not require antibodies

- Capable of detecting weak and transient protein interactions

- In situ labeling of proteins proximal to the target protein within live cells