Proteins are indispensable for life due to their versatility and specificity. They are involved in virtually every cellular process from catalytic reactions, structural integrity, signaling and communication to immune defense and pathogenicity.

Their ability to adopt diverse structures and functions makes them central to the complexity and adaptability of all living organisms. As they usually interact with other proteins or cellular components to carry out complex biological processes they rarely function alone. Thus, identifying Protein-Protein-Interactions (PPIs) helps elucidating the complex networks and pathways that underlie cellular functions and organismal biology.

Methods for studying PPIs include yeast two-hybrid screening, co-immunoprecipitation, mass spectrometry, fluorescence resonance energy transfer, computational approaches and many others.

Although each method has its specific advantages, they all have disadvantages such as a high false positive rate, a high technical complexity and low efficiency, which makes precise identifications of specific interaction partners somtimes difficult.

Here we present our TIE-UP-SIN method which represents a Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization.

This method provides an in vivo approach for the identification of PPIs based on stable isotope labeling, reversible crosslinking and affinity purification coupled with highly sensitive PPI identification by mass spectrometry (MS). The isotope labeling is essential for normalizing MS sample data as it addresses variability, ensures accurate quantification, and enhances the reliability of comparative DIA-MS proteomic analyses. This leads to precise and reproducible results, facilitating robust biological insights eliminating high false discovery rates. The combination with a quick and cheap reversible crosslinking and affinity purification protocol makes this technique a powerful tool to stabilize transient or weak in vivo interactions and to specifically capture them in a highly specific purification process.

Overall, TIE-UP-SIN is a robust and favorably priced method for studying PPIs, offering quick and reliable insights into the composition and architecture of protein complexes in a biological in vivo context. The utility of the approach will be presented using the well-described and essential housekeeping sigma factor SigA from Bacillus subtilis as an example.