biostudies-literatureUnknown16Schedlowski MProteins function through complex interaction networks that govern nearly all aspects of cellular physiology. Identifying protein-protein interactions (PPIs) under native conditions remains challenging due to the transient nature of many complexes and technical limitations of conventional approaches. We present TIE-UP-SIN (Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization), a robust and reproducible method for <i>in vivo</i> identification of PPIs. This approach combines metabolic labeling with ¹⁵N isotopes, reversible <i>in vivo</i> formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN is specifically designed to preserve transient or weak interactions during purification and to quantify interaction pFrontiers in microbiology1657647https://www.ebi.ac.uk/biostudies/studies/S-EPMC12434083biostudies-literatureTIE-UP-SIN: a novel method for enhanced identification of protein-protein interactions.PMC12434083Salazar MGSchedlowski MSteil LSurmann KReder AHentschker CHarms MHoffmuller TVolker UMichalik SfalseTIE-UP-SIN: a novel method for enhanced identification of protein-protein interactions.Proteins function through complex interaction networks that govern nearly all aspects of cellular physiology. Identifying protein-protein interactions (PPIs) under native conditions remains challenging due to the transient nature of many complexes and technical limitations of conventional approaches. We present TIE-UP-SIN (Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization), a robust and reproducible method for <i>in vivo</i> identification of PPIs. This approach combines metabolic labeling with ¹⁵N isotopes, reversible <i>in vivo</i> formaldehyde crosslinking, affinity purification, and quantitative mass spectrometry. TIE-UP-SIN is specifically designed to preserve transient or weak interactions during purification and to quantify interaction p2025-01-01T00:00:00Z20252026-06-03T02:54:15.165Z2026-04-23T03:13:08.706ZS-EPMC124340834095922910.3389/fmicb.2025.1657647