Louis Delhaye, George D Moschonas, Daria Fijalkowska, Annick Verhee, Delphine De Sutter, Tessa Van de Steene, Margaux De Meyer, Hanna Grzesik, Laura Van Moortel, Karolien De Bosscher, Thomas Jacobs, Sven Eyckerman
{"title":"Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.","authors":"Louis Delhaye, George D Moschonas, Daria Fijalkowska, Annick Verhee, Delphine De Sutter, Tessa Van de Steene, Margaux De Meyer, Hanna Grzesik, Laura Van Moortel, Karolien De Bosscher, Thomas Jacobs, Sven Eyckerman","doi":"10.1016/j.crmeth.2024.100818","DOIUrl":null,"url":null,"abstract":"<p><p>Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.</p>","PeriodicalId":29773,"journal":{"name":"Cell Reports Methods","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11294833/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell Reports Methods","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1016/j.crmeth.2024.100818","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/9 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Protein-protein interactions play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spectrometry-based proteomics overcomes challenges typically associated with other methods and has quickly become the current state of the art in the field. Nevertheless, tight control of proximity-labeling enzymatic activity and expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleaving mutant to accommodate the protein of interest in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we built a Golden Gate modular cloning system to generate plasmids for transient expression and stable integration. To highlight our T2A Split/link design, we applied it to identify protein interactions of the glucocorticoid receptor and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and non-structural protein 7 (NSP7) proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control that builds upon previously established control requirements in the field.