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

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.

SUBMITTER: Luo S 

PROVIDER: S-EPMC12539243 | biostudies-literature | 2025 Sep

REPOSITORIES: biostudies-literature