Streamlined Biotinylation, Enrichment and Analysis for Enhanced Plasma Membrane Protein Identification Using Turboid and Turboid-Start Biotin Ligases

Abstract

Abstract Plasma membrane proteins play pivotal roles in various cellular events and are crucial in disease pathogenesis, making their comprehensive characterization vital for biomedical research. However, the hydrophobic nature and low expression levels of PMPs pose challenges for conventional enrichment methods, hindering their identification and functional profiling. In this study, we presented a novel TurboID-based enrichment approach for PMPs that helped overcoming some of the existing limitations. We evaluated the efficacy of TurboID and its modified form, TurboID-START, in PMP enrichment, achieving more efficient and targeted labeling of PMPs without the need for stable cell line generation. This approach significantly reduced non-specific biotinylation events, leading to improved PMP enrichment and enabling a precise assessment of the subcellular proteome associated with the plasma membrane. Our findings highlighted the dynamic nature of the plasma membrane as well and captured transient associations of proteins with the plasma membrane. The novel TurboID-based enrichment approach presented here offers promising prospects for in-depth investigations into PMPs and their roles in cellular processes.