Exploring Protein Interactomes Using TurboID-Directed Proximity Labeling and Mass Spectrometry

Area of Science:

• Biochemistry
• Cell Biology
• Parasitology

Background:

• Traditional protein interaction mapping methods struggle with transient interactions.
• Proximity labeling (PL) offers a solution for capturing dynamic protein associations in vivo.
• Trypanosoma cruzi interactions are crucial for understanding parasitic diseases.

Purpose of the Study:

• To develop and standardize a proximity labeling protocol for Trypanosoma cruzi.
• To create a novel vector system (pTcTurboID) for stable and regulatable protein expression.
• To generate high-confidence proximity proteomes in T. cruzi.

Main Methods:

• Utilized a novel vector system, pTcTurboID, for TurboID fusion protein expression.
• Implemented an eight-step protocol including lineage generation, biotinylation optimization, and streptavidin bead purification.
• Employed compartment-specific spatial controls and statistical analysis to identify true interactors.

Main Results:

• Successfully generated reproducible proximity interactomes using nuclear and cytoplasmic baits.
• Validated the pTcTurboID system for stable and regulatable bait expression.
• Demonstrated minimal bait expression to preserve physiological function while ensuring biotinylation activity.

Conclusions:

• The developed protocol offers an efficient, reproducible, and robust framework for proximity proteomics in T. cruzi.
• This method advances the study of protein interactions in this important parasite.
• Enables high-confidence mapping of both stable and transient protein interactions.