APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Peroxidase-catalyzed proximity labeling is a key technique for in vivo protein interaction studies.
• APEX2 enzyme fusions enable the labeling of proteins in a protein's immediate vicinity.
• This method captures a snapshot of the local protein environment.

Purpose of the Study:

• To describe a robust pipeline for proximity-labeled protein enrichment and analysis.
• To enable the study of dynamic protein interaction changes in response to perturbations.
• To facilitate comparative analyses of protein proximity in different biological contexts.

Main Methods:

• Utilizing APEX2 for proximity-dependent biotinylation of nearby proteins.
• Employing denaturing purification with magnetic streptavidin beads.
• Implementing multiplexed quantitative mass spectrometry, including TMT labeling, peptide fractionation, and SPS MS3 analysis.

Main Results:

• Successful enrichment of proximity-labeled proteins under stringent denaturing conditions.
• A comprehensive workflow for sample preparation for quantitative mass spectrometry.
• Demonstrated capability for parallel quantification of hundreds of proteins across multiple conditions and time points.

Conclusions:

• The described method provides a powerful tool for investigating complex protein environments.
• This approach is suitable for studying dynamic changes in protein proximity in perturbed systems.
• Multiplexed quantitative mass spectrometry enhances the capacity for high-throughput proteomic analysis of protein interactions.