TIE-UP-SIN: A Method for Enhanced Identification of Protein-Protein Interactions.

Protein-protein interactions (PPIs) govern nearly all aspects of cellular physiology, yet identifying these interactions under native conditions remains challenging. Here, we present TIE-UP-SIN (targeted interactome experiment for unknown proteins by stable isotope normalization), a robust method for in vivo identification and quantification of PPIs in bacterial systems. The protocol combines metabolic labeling with 15N isotopes, reversible formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN preserves transient or weak interactions during purification and quantifies interaction partners using internal light/heavy peptide ratios, reducing experimental variability. The method employs a triple-sample design to distinguish specific from nonspecific interactors and can be adapted to various bacterial species and affinity tags. Data analysis is streamlined through a user-friendly web application (https://shiny-fungene.biologie.uni-greifswald.de/TIE_UP_SIN_app) that automates statistical analysis, normalization, and visualization, requiring no programming expertise. The entire workflow from cell culture to mass spectrometry data acquisition takes approximately 4-5 days, with data analysis completed in 1-2 days using the web application. Key features • Captures transient protein interactions in vivo through reversible formaldehyde crosslinking under native expression conditions. • Internal 15N metabolic labeling enables robust quantification and reduces experimental variability across biological replicates. • Triple-sample design (WT/WT, bait/WT, bait/bait) distinguishes specific from nonspecific interactors with high confidence. • Applicable to diverse bacterial systems with simple adaptation to any affinity-tagged bait protein.