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.