Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling
Abstract 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...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2024-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-024-83928-w |
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| author | Zora Chui-Kuen Chan Cheng Qi Yuanhong Cai Xin Li Jing Ren |
| author_facet | Zora Chui-Kuen Chan Cheng Qi Yuanhong Cai Xin Li Jing Ren |
| author_sort | Zora Chui-Kuen Chan |
| collection | DOAJ |
| description | Abstract 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. |
| format | Article |
| id | doaj-art-8d56b37bcff8459e8ddd057ecd47062f |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-8d56b37bcff8459e8ddd057ecd47062f2025-01-05T12:29:39ZengNature PortfolioScientific Reports2045-23222024-12-0114111210.1038/s41598-024-83928-wRevealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labellingZora Chui-Kuen Chan0Cheng Qi1Yuanhong Cai2Xin Li3Jing Ren4Neurobiology Division, MRC Laboratory of Molecular BiologyNeurobiology Division, MRC Laboratory of Molecular BiologyInstitute of Chemical Biology, Shenzhen Bay LaboratoryInstitute of Chemical Biology, Shenzhen Bay LaboratoryNeurobiology Division, MRC Laboratory of Molecular BiologyAbstract 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.https://doi.org/10.1038/s41598-024-83928-w |
| spellingShingle | Zora Chui-Kuen Chan Cheng Qi Yuanhong Cai Xin Li Jing Ren Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling Scientific Reports |
| title | Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling |
| title_full | Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling |
| title_fullStr | Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling |
| title_full_unstemmed | Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling |
| title_short | Revealing and mitigating the inhibitory effect of serotonin on HRP-mediated protein labelling |
| title_sort | revealing and mitigating the inhibitory effect of serotonin on hrp mediated protein labelling |
| url | https://doi.org/10.1038/s41598-024-83928-w |
| work_keys_str_mv | AT zorachuikuenchan revealingandmitigatingtheinhibitoryeffectofserotoninonhrpmediatedproteinlabelling AT chengqi revealingandmitigatingtheinhibitoryeffectofserotoninonhrpmediatedproteinlabelling AT yuanhongcai revealingandmitigatingtheinhibitoryeffectofserotoninonhrpmediatedproteinlabelling AT xinli revealingandmitigatingtheinhibitoryeffectofserotoninonhrpmediatedproteinlabelling AT jingren revealingandmitigatingtheinhibitoryeffectofserotoninonhrpmediatedproteinlabelling |