Dataset Information

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

ABSTRACT: Proteins are indispensable biomolecules playing vital roles in nearly every aspect of cellular function. They participate in processes such as enzymatic catalysis, signaling pathways, cell responses, pathogenicity as well as structural components of cells. Their structural and functional diversity are the foundation of biological complexity and adaptability. As many proteins function collaboratively through interactions with others, identifying protein-protein interactions (PPIs) is fundamental to understanding the complicated networks and pathways governing cellular activities. Numerous methods exist to study PPIs, including two-hybrid screening, co-immunoprecipitation, mass spectrometry (MS), and computational approaches. While each technique has its strengths, limitations such as high false-positive rates, technical complexity, and low efficiency can hinder precise identification of specific interaction partners. We present TIE-UP-SIN (Targeted Interactome Experiment for Unknown Proteins by Stable Isotope Normalization), a novel simple and straight forward approach for in vivo PPI identification. It combines stable isotope labeling, reversible crosslinking, and affinity purification with highly sensitive mass spectrometry analysis. Stable isotope labeling improves MS data by enhancing quantification accuracy and reproducibility while reducing the number of false discoveries. Rapid, cost-effective reversible crosslinking captures transient interactions for stringent purification, ensuring maximum specificity. The effectiveness of TIE-UP-SIN is showcased by identifying key interaction partners of the essential Bacillus subtilis sigma factor RpoD (SigA) under control and ethanol stress conditions, highlighting its reliability and affordability for in vivo protein-protein interaction studies.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Bacillus Subtilis (ncbitaxon:1423)

SUBMITTER: Alexander Reder