Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling

Proximity-dependent biotinylation coupled with mass spectrometry enables the characterization of subcellular proteomes. This technique has significantly advanced neuroscience by revealing sub-synaptic protein networks, such as the synaptic cleft and post-synaptic density. Profiling proteins at this detailed level is essential for understanding the molecular mechanisms of neuronal connectivity and transmission. Despite its recent successful application to various neuronal types, proximity labelling has yet to be employed to study the serotonin system. In this study, we uncovered an unreported inhibitory mechanism of serotonin on horseradish peroxidase (HRP)-based biotinylation. Our result showed that serotonin significantly reduces biotinylation levels across various Biotin-XX-tyramide (BxxP) concentrations in HEK293T cells and primary neurons, whereas dopamine exerts minimal interference, highlighting the specificity of this inhibition. To counteract this inhibition, we demonstrated that Dz-PEG, an aryl diazonium compound that consumes serotonin through an azo-coupling reaction, restores biotinylation efficiency. Label-free quantitative proteomics confirmed that serotonin inhibits biotinylation, and that Dz-PEG effectively reverses this inhibition. These findings highlight the importance of accounting for neurotransmitter interference in proximity-dependent biotinylation studies, especially for cell-type specific profiling in neuroscience. Additionally, we provided a potential strategy to mitigate these challenges, thereby enhancing the accuracy and reliability of such studies.