Back to Search Start Over

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
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.

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