Leveraging a self-cleaving peptide for tailored control in ...

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Highlights

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  We present a strategy to expand BioID-only controls in proximity labeling experiments

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  We introduce self-cleaving and mutant T2A linkers for differential proximity labeling

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  MUTT2A/T2A differential analysis efficiently filters common BioID contaminants

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  We present a rapid and customizable plasmid assembly platform for proximity labeling

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.

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.

Keyword(s)

proximity labeling

T2A peptide

glucocorticoid receptor

nucleocapsid protein

non-structural protein 7

Golden Gate assembly

TurboID

proteomics

interactomics

T2A split/link

Research topic(s)

CP: Biotechnology

© 2024 The Authors. Published by Elsevier Inc.