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.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health challenge, primarily due to the increasing prevalence of drug resistance. Consequently, the development of drugs with novel modes of action (MOAs) is urgently required. In this study, we discovered and characterized two potent inhibitors, Pi-1-58 and Pi-2-26, targeting the prokaryotic ubiquitin-like protein (Pup) ligase PafA of Mtb. Pi-1-58 was identified through computer-aided drug screening. The binding mode of Pi-1-58 and PafA was investigated through molecular docking, followed by experimental validations. Based on the core structure of Pi-1-58, we developed a more potent inhibitor, Pi-2-26, through structure-guided drug design. Both Pi-1-58 and Pi-2-26 exhibited selective and specific inhibition of PafA according to biochemical and cell-based assays. Importantly, the inhibitors demonstrated significant inhibition on Mtb survival in the presence of nitric oxide, mimicking the in vivo nitrogen limited environment that Mtb encountered in macrophage. Our findings provide a comprehensive understanding of the structural and functional aspects of these PafA inhibitors and establish a solid foundation for the development of novel therapeutics against tuberculosis.

The stomach harbors a complex microbial niche, yet the persistence and disease relevance of its core microbiota remain unclear. Using full-length 16S and ITS sequencing of 255 pediatric samples, this study identifies 11 core bacterial genera organized into two antagonistic communities, CST1A and CST1B. CST1A is enriched in non-ulcer controls, whereas CST1B is associated with gastroduodenal disease, with limited fungal involvement. Stratified analyses show that Helicobacter pylori infection depletes CST1A taxa. Functional profiling indicates that CST1A is linked to motility-related processes, while CST1B favors nucleotide and peptidoglycan biosynthesis. In vitro and in vivo experiments demonstrate that CST1A-derived Achromobacter spanius alleviates gastric mucosal injury by modulating inflammatory responses. Cross-cohort validation across eight datasets (n = 1,299) confirms the persistent relevance of the CST1A-CST1B structure across disease stages. Together, these findings define an early-life core gastric microbiota linked to disease risk and conserved across the lifespan.

SUMMARY: 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 an innovative proteomic method termed A ffinity P urification coupled P roximity L ab E ling-Mass Spectrometry (APPLE-MS), which combines the high specificity of Twin-Strep-tag enrichment with PafA-mediated proximity labeling. This method achieves unprecedented sensitivity while maintaining high specificity (4.07-fold over AP-MS). APPLE-MS also revealed the dynamic mitochondrial interactome of 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. GRAPHICAL MOTIVATION – Cell Reports Methods only: 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. SIGNIFICANCE – Cell Chemical Biology only: Proximity labeling is a powerful tool in chemical biology, providing critical support for studying protein function, cell signaling, and disease mechanisms by chemically labeling and capturing biomolecular interactions. In this study, we introduce a modular affinity purification-mass spectrometry (AP-MS) method that integrates proximity labeling. This method leverages the proximity-dependent enzymatic activity of PafA to covalently attach Pup ^E to nearby proteins, in combination with the high-affinity interaction between Twin-Strep-tag and streptavidin to achieve efficient binding. This innovative approach enables precise identification of protein-protein interactions (PPIs), offering a powerful tool for capturing weak and transient interactions that are often missed by traditional AP-MS techniques. By enhancing both the specificity and efficiency of protein labeling, our work provides a robust chemical biology strategy to elucidate complex protein interactions, which are essential for understanding cellular processes and developing targeted therapeutic strategies, offering new insights into protein function and interaction dynamics in both health and disease.