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Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics
- Source :
- Cell Reports: Methods, Vol 4, Iss 7, Pp 100818- (2024)
- Publication Year :
- 2024
- Publisher :
- Elsevier, 2024.
-
Abstract
- Summary: 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. Motivation: In proximity labeling proteomics, protein-protein interactions are identified by in vivo biotinylation. However, the current lack of a universally applicable negative control for differential analysis affects accurate mapping of the interactome. To bridge this gap, we conceptualized a system based on the T2A self-cleaving peptide to match expression levels between control and bait protein setups while using the same bait protein. In addition, we implemented a versatile modular cloning system to build mammalian expression vectors for, but not limited to, proximity labeling assays.
- Subjects :
- CP: Biotechnology
Biotechnology
TP248.13-248.65
Biochemistry
QD415-436
Science
Subjects
Details
- Language :
- English
- ISSN :
- 26672375
- Volume :
- 4
- Issue :
- 7
- Database :
- Directory of Open Access Journals
- Journal :
- Cell Reports: Methods
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.23617f47cc47455eb8fa9874ec8082a9
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.crmeth.2024.100818