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

## TL;DR

APPLE-MS is a new method that improves the detection of protein interactions, especially weak and membrane-associated ones, using a combination of affinity purification and proximity labeling.

## Contribution

APPLE-MS integrates Twin-Strep tag enrichment and PafA-mediated proximity labeling to enhance PPI detection with high specificity and sensitivity.

## Key findings

- APPLE-MS reveals the dynamic mitochondrial interactome of SARS-CoV-2 ORF9B during antiviral responses.
- APPLE-MS enables in situ mapping of GLP-1 receptor complexes, showing its utility for membrane PPI studies.
- Endogenous PIN1 profiling with APPLE-MS uncovers novel roles in DNA replication.

## Abstract

While affinity purification-mass spectrometry (AP-MS) has significantly advanced protein-protein interaction (PPI) studies, its limitations in detecting weak, transient, and membrane-associated interactions remain. To address these challenges, we introduced a proteomic method termed affinity purification coupled proximity labeling-mass spectrometry (APPLE-MS), which combines the high specificity of Twin-Strep tag enrichment with PafA-mediated proximity labeling. This method achieves improved sensitivity while maintaining high specificity (4.07-fold over AP-MS). APPLE-MS also revealed the dynamic mitochondrial interactome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ORF9B during antiviral responses, while endogenous PIN1 profiling uncovered novel roles in DNA replication. Notably, APPLE-MS enabled in situ mapping of GLP-1 receptor complexes, demonstrating its unique capabilities for membrane PPI studies. This versatile method advances interactome research by providing comprehensive, physiologically relevant PPI networks, opening new opportunities for mechanistic discovery and therapeutic targeting.

•Combined Twin-Strep tag enrichment and PafA-mediated labeling enhances PPI detection•APPLE-MS captures weak/transient interactions and membrane PPIs in native contexts•APPLE-MS reveals dynamic SARS-CoV-2 ORF9B interactome during antiviral responses•APPLE-MS enables endogenous PIN1 interactome mapping with minimal tag interference

Combined Twin-Strep tag enrichment and PafA-mediated labeling enhances PPI detection

APPLE-MS captures weak/transient interactions and membrane PPIs in native contexts

APPLE-MS reveals dynamic SARS-CoV-2 ORF9B interactome during antiviral responses

APPLE-MS enables endogenous PIN1 interactome mapping with minimal tag interference

Affinity purification-mass spectrometry (AP-MS) has become a widely used method for capturing high-affinity protein-protein interactions (PPIs). However, it still faces several limitations, including high levels of non-specific binding, challenges in detecting weak interactions, and limited ability to identify PPIs at the cell surface in situ. To overcome these challenges, we introduce a modular AP-MS method enhanced by PafA-mediated proximity labeling, which improves both the specificity and sensitivity of PPI detection in a single, streamlined workflow.

Luo et al. introduce APPLE-MS, a method integrating affinity purification and PafA-mediated proximity labeling to overcome limitations in detecting weak, transient, and membrane-associated protein interactions. The approach reveals dynamic viral-host interactomes and endogenous protein networks, offering broad applications in interactome research and therapeutic target discovery.