Inhibitory effect of serotonin on HRP-based proximity labelling

The application of biotin ligase or peroxidase fused with specific proteins or subcellular regions, combined with mass spectrometry-based quantitative proteomics, has become a potent approach for unravelling molecular interactions and determining the locations of native proteins based on their proximity. Recent applications of proximity-dependent biotinylation in neuroscience have yielded insights into the composition of sub-synaptic structures, including the synaptic cleft [1].Despite the effectiveness of proximity labelling methods in providing a global perspective on protein-protein interactions, challenges emerged when horseradish peroxidase (HRP)-based proximity labelling was directly applied to the serotonin system. Our findings uncover a previously unreported inhibitory mechanism of serotonin on HRP-based biotinylation. Elevated serotonin concentrations were observed to impede the efficient biotinylation facilitated by HRP expressed on the surface of HEK293T cells. To overcome this hindrance, we employed an azo-coupling reaction to block the hydroxyl group (−OH) of released serotonin, allowing proximity labelling in the presence of serotonin. Notably, this inhibitory phenomenon was consistently demonstrated in primary neuronal cultures, underscoring its biological relevance in neurons.In conclusion, our study revealed the mechanism behind the limited application of HRP-based proximity labelling in the serotonin system and provided a potential solution to overcome this problem. The implication of this study lies in the enhanced applicability of proximity labelling techniques to investigate the complex interplay of proteins within the serotonin system.1. Han, S., J. Li, and A.Y. Ting, Proximity labeling: spatially resolved proteomic mapping for neurobiology. Current Opinion in Neurobiology, 2018. 50: p. 17-23.