%0 Journal Article
%T Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics.
%A Delhaye L
%A Moschonas GD
%A Fijalkowska D
%A Verhee A
%A De Sutter D
%A Van de Steene T
%A De Meyer M
%A Grzesik H
%A Van Moortel L
%A De Bosscher K
%A Jacobs T
%A Eyckerman S
%J Cell Rep Methods
%V 4
%N 7
%D 2024 Jul 15
%M 38986614
暂无%R 10.1016/j.crmeth.2024.100818
%X 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.