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

ABSTRACT: 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 allow the use of the protein-of-interest 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 protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control to match expression levels for proximity labeling proteomics.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Louis Delhaye

LAB HEAD: Sven Eyckerman

PROVIDER: PXD046413 | Pride | 2024-06-10

REPOSITORIES: Pride

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			Action	DRS
	B102794_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-183-1.raw	Raw
	B102794_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-183-2.raw	Raw
	B102796_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-184-1.raw	Raw
	B102796_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-184-2.raw	Raw
	B102804_Ap_110uPAc_LumosLC_CMB-1046_SVEYC_AP-MS-183-3.raw	Raw

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

Project description: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 allow the use of the protein-of-interest 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 protein interactions of the glucocorticoid receptor and SARS-CoV-2 nucleocapsid and NSP7 proteins by TurboID proximity labeling. Our results demonstrate that our T2A split/link provides an opportune control to match expression levels for proximity labeling proteomics.

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

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