Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM
Protein O-GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, O-GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation,...
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Bio-protocol LLC
2025-03-01
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| author | Qin Lei Haibin Yu Fang Chen Kai Yuan |
| author_facet | Qin Lei Haibin Yu Fang Chen Kai Yuan |
| author_sort | Qin Lei |
| collection | DOAJ |
| description | Protein O-GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, O-GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation, and protein homeostasis. Dysregulation of O-GlcNAc homeostasis has been implicated in a variety of diseases, including cardiovascular diseases, cancer, and neurodegenerative diseases. Studying O-GlcNAcylated proteins in different tissues is crucial to understanding the pathogenesis of these diseases. However, identifying phenotype-relevant candidate substrates in a tissue-specific manner remains unfeasible. We developed a novel tool for the analysis of O-GlcNAcylated proteins, combining a catalytically inactive CpOGA mutant CpOGACD and TurboID proximity labeling technology. This tool converts O-GlcNAc modifications into biotin labeling, enabling the enrichment and mass spectrometry (MS) identification of O-GlcNAcylated proteins in specific tissues. Meanwhile, TurboID-CpOGADM, which carries two point mutations that inactivate both its catalytic and binding activities toward O-GlcNAc modification, was used as a control to differentiate O-GlcNAc-independent protein–protein interactions. We have successfully used TurboID-CpOGACD/DM (TurboID-CpOGAM) to enrich O-GlcNAc proteins in Drosophila combining the UAS/Gal4 system. Our protocol provides a comprehensive workflow for tissue-specific enrichment of candidate O-GlcNAcylated substrates and offers a valuable tool for dissecting tissue-specific O-GlcNAcylation functions in Drosophila. |
| format | Article |
| id | doaj-art-1e7cef2d86bd4e8588485f6ebed6824e |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Bio-protocol LLC |
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| spelling | doaj-art-1e7cef2d86bd4e8588485f6ebed6824e2025-08-20T02:02:05ZengBio-protocol LLCBio-Protocol2331-83252025-03-0115510.21769/BioProtoc.5234Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAMQin Lei0Haibin Yu1Fang Chen2Kai Yuan3Hunan Key Laboratory of Molecular Precision Medicine, Department of Oncology, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Molecular Precision Medicine, Department of Oncology, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Molecular Precision Medicine, Department of Oncology, Xiangya Hospital, Central South University, Changsha, ChinaHunan Key Laboratory of Molecular Precision Medicine, Department of Oncology, Xiangya Hospital, Central South University, Changsha, ChinaCenter for Medical Genetics, School of Life Sciences, Central South University, Changsha, China, Furong Laboratory, Changsha, China, National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha, China, The Biobank of Xiangya Hospital, Central South University, Changsha, ChinaProtein O-GlcNAcylation is a prevalent and dynamic post-translational modification that targets a multitude of nuclear and cytoplasmic proteins. Through the modification of diverse substrates, O-GlcNAcylation plays a pivotal role in essential cellular processes, including transcription, translation, and protein homeostasis. Dysregulation of O-GlcNAc homeostasis has been implicated in a variety of diseases, including cardiovascular diseases, cancer, and neurodegenerative diseases. Studying O-GlcNAcylated proteins in different tissues is crucial to understanding the pathogenesis of these diseases. However, identifying phenotype-relevant candidate substrates in a tissue-specific manner remains unfeasible. We developed a novel tool for the analysis of O-GlcNAcylated proteins, combining a catalytically inactive CpOGA mutant CpOGACD and TurboID proximity labeling technology. This tool converts O-GlcNAc modifications into biotin labeling, enabling the enrichment and mass spectrometry (MS) identification of O-GlcNAcylated proteins in specific tissues. Meanwhile, TurboID-CpOGADM, which carries two point mutations that inactivate both its catalytic and binding activities toward O-GlcNAc modification, was used as a control to differentiate O-GlcNAc-independent protein–protein interactions. We have successfully used TurboID-CpOGACD/DM (TurboID-CpOGAM) to enrich O-GlcNAc proteins in Drosophila combining the UAS/Gal4 system. Our protocol provides a comprehensive workflow for tissue-specific enrichment of candidate O-GlcNAcylated substrates and offers a valuable tool for dissecting tissue-specific O-GlcNAcylation functions in Drosophila.https://bio-protocol.org/en/bpdetail?id=5234&type=0 |
| spellingShingle | Qin Lei Haibin Yu Fang Chen Kai Yuan Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM Bio-Protocol |
| title | Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM |
| title_full | Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM |
| title_fullStr | Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM |
| title_full_unstemmed | Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM |
| title_short | Tissue-Specific Profiling of O-GlcNAcylated Proteins in Drosophila Using TurboID-CpOGAM |
| title_sort | tissue specific profiling of o glcnacylated proteins in drosophila using turboid cpogam |
| url | https://bio-protocol.org/en/bpdetail?id=5234&type=0 |
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