Sensitive and specific affinity purification-mass spectrometry assisted by PafA-mediated proximity labeling

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• Affinity purification-mass spectrometry (AP-MS) is crucial for protein-protein interaction (PPI) studies.
• Existing AP-MS methods struggle with weak, transient, and membrane-associated interactions.

Purpose of the Study:

• To develop an advanced proteomic method for sensitive and specific detection of PPIs.
• To overcome limitations of traditional AP-MS for complex biological systems.

Main Methods:

• Introduced Affinity Purification coupled Proximity Labeling-Mass Spectrometry (APPLE-MS).
• Combined Twin-Strep tag enrichment with PafA-mediated proximity labeling.
• Applied APPLE-MS to study SARS-CoV-2 ORF9B mitochondrial interactome, endogenous PIN1, and GLP-1 receptor complexes.

Main Results:

• APPLE-MS demonstrated significantly improved sensitivity and specificity (4.07-fold over AP-MS).
• Revealed dynamic mitochondrial interactome of SARS-CoV-2 ORF9B during antiviral responses.
• Uncovered novel roles for PIN1 in DNA replication and enabled in situ mapping of membrane protein complexes.

Conclusions:

• APPLE-MS is a versatile proteomic tool for comprehensive interactome mapping.
• The method provides physiologically relevant PPI networks, aiding mechanistic discovery.
• Opens new avenues for therapeutic target identification, particularly for membrane proteins.