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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

Project description: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.

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Project description:Accumulating evidence highlights the role of long non-coding RNAs (lncRNA) in cellular homeostasis, and their dysregulation in disease settings. Most lncRNAs function by interacting with proteins or protein complexes. While several orthogonal methods have been developed to identify these proteins, each method has its inherent strengths and limitations. Here, we combine two RNA-centric methods ChIRP-MS and RNA-BioID to obtain a comprehensive list of proteins that interact with the well-known lncRNA HOTAIR. Overexpression of HOTAIR has been associated with a metastasis-promoting phenotype in various cancers. Although HOTAIR is known to bind with PRC2 and LSD1 protein complexes, an unbiased and comprehensive method to map its interactome has not yet been performed. Both ChIRP-MS and RNA-BioID data sets show an association of HOTAIR with mitoribosomes, suggesting HOTAIR has functions independent of its (post-)transcriptional mode-of-action.

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