Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics

ABSTRACT: PPIs play an important biological role in every aspect of cellular homeostasis and functioning. Proximity labeling mass spec-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 acitivty a,d expression levels is crucial to accurately identify protein interactors. Here, we leverage a T2A self-cleaving peptide and a non-cleavage mutant to allow the use of the POI in the experimental and control TurboID setup. To allow easy and streamlined plasmid assembly, we build 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 PPIs of GR, NCAP, and NSP7 by TurboID proximity labeling proteomics. Our results demisntrate that our T2A split-link provides an opportune control that builds upon previously established control requirements in the field.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Louis Delhaye  

LAB HEAD: Sven Eyckerman

PROVIDER: PXD046412 | Pride | 2024-06-10

REPOSITORIES: Pride