TurboID Labeling and Analysis of Proteins in the Primary Cilium.

Known as the cell's antenna and signaling hub, the primary cilium is a hair-like organelle with a few micrometers in length and 200-300 nm in diameter. Due to the small size of the primary cilium, it is technically challenging to profile ciliary proteins from mammalian cells. Traditional methods, such as physical isolation of cilia, are susceptible to contamination from other cellular components. Other proximity-based labeling methods via APEX or BioID have been used to map ciliary proteins. However, these approaches have their inherent limitations, including the use of toxic reagents like H₂O₂ and prolonged labeling kinetics. Here, we show a new proximity-based labeling technique for primary cilia with TurboID. TurboID presents a distinct advantage over BioID and APEX2 due to its expedited labeling kinetics, taking minutes instead of hours, and its use of a non-toxic biotin substrate, which eliminates the need for H₂O₂. When targeted to the cilium, TurboID selectively labels ciliary proteins with biotin. The biotinylated proteins are then enriched with streptavidin beads and labeled with tandem mass tags (TMT), followed by mass spectrometry (MS) detection. This protocol eliminates the requirement of toxic labeling reagents and significantly reduces the labeling time, thus providing advantages in mapping signaling proteins with high temporal resolution in live cells. Key features • Compared to other proximity labeling enzymes, TurboID offers fast labeling kinetics and uses cell-permeable biotin as the labeling reagent [1]. • This protocol includes a straightforward subcellular fractionation step to remove the nuclei to reduce the non-specific background. • This protocol has been successfully applied to the NIH 3T3 cell line and could also be applied in other cell lines and animal tissues.