Investigating the interactomic landscape of survival motor neuron (SMN) and the SMNΔ7 truncated protein.

Abstract

This protocol describes a methodology combining TurboID - a recently developed proximity biotinylation technique - with conventional epitope-tag based co-immunoprecipitation (Co-IP) to analyze protein-protein interactions (PPIs) in cell culture systems. This integrated approach allows the targeted examination of both transient and stable protein interactors, enhancing our understanding of protein dynamics. TurboID captures transient interactions often missed by Co-IP, which captures high affinity, stable interactors. Combination of both techniques enables direct comparison of interaction strengths, providing insights into the dynamic nature of protein interactions within cells. The rapid biotinylation capability of TurboID reduces background noise and false positives while Co-IP enriches stable interactors, together improving data quality and interpretation of the interactomic landscape. Demonstrating the efficacy of this methodology, proteins relevant to the pathology of Spinal Muscular Atrophy were utilized to explore variations at the interactome level. The use of identical starting cell lysates for both TurboID and Co-IP minimized variability and ensured datasets were comparable, allowing for consistency and enhancing the reliability of findings regarding the nature and strength of protein interactions. This novel framework effectively combines both innovative and classical techniques while maintaining consistency in sample handling, advancing our understanding of the intricate networks that govern cellular processes.

Full data-set available at https://www.ebi.ac.uk/pride/ reference ID PXD066858.