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

Plasma membrane proteins (PMPs) 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 efficient and targeted labelling of PMPs without the need for stable cell line generation. This approach resulted reduction in non-specific biotinylation events, leading to improved PMP enrichment and enabled assessment of the subcellular proteome associated with the plasma membrane. Our findings paved the way for studies targeting the dynamic nature of the plasma membrane proteome and aiming to capture 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. Graphical Abstract Graphical presentation of the experimental work-flow used in this study. TurboID and TurboID-START enzymes were produced in Escherichia coli , purified to homogeneity and added onto the cultured CHO cells to carry out PMP biotinylation. The biotinylated proteins were then enriched using streptavidin-coated beads before on-bead tryptic digestions were performed. At the final stage, a comprehensive LC–MS/MS analysis was performed for protein identification.