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

Louis Delhaye (UGent) , Georgios Moschonas (UGent) , Daria Fijalkowska (UGent) , Annick Verhee (UGent) , Delphine De Sutter (UGent) , Tessa Van de Steene (UGent) , Margaux De Meyer (UGent) , Hanna Grzesik (UGent) , Laura Van Moortel (UGent) , Karolien De Bosscher (UGent) , et al.
(2024) CELL REPORTS METHODS. 4(7).
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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 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.
Keywords
GLUCOCORTICOID-RECEPTOR, PRECISION, MECHANISM, PROTEINS

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MLA
Delhaye, Louis, et al. “Leveraging a Self-Cleaving Peptide for Tailored Control in Proximity Labeling Proteomics.” CELL REPORTS METHODS, vol. 4, no. 7, Cell Press, 2024, p. 100818, doi:10.1016/j.crmeth.2024.100818.
APA
Delhaye, L., Moschonas, G., Fijalkowska, D., Verhee, A., De Sutter, D., Van de Steene, T., … Eyckerman, S. (2024). Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics. CELL REPORTS METHODS, 4(7), 100818. https://doi.org/10.1016/j.crmeth.2024.100818
Chicago author-date
Delhaye, Louis, Georgios Moschonas, Daria Fijalkowska, Annick Verhee, Delphine De Sutter, Tessa Van de Steene, Margaux De Meyer, et al. 2024. “Leveraging a Self-Cleaving Peptide for Tailored Control in Proximity Labeling Proteomics.” CELL REPORTS METHODS 4 (7): 100818. https://doi.org/10.1016/j.crmeth.2024.100818.
Chicago author-date (all authors)
Delhaye, Louis, Georgios Moschonas, Daria Fijalkowska, Annick Verhee, Delphine De Sutter, Tessa Van de Steene, Margaux De Meyer, Hanna Grzesik, Laura Van Moortel, Karolien De Bosscher, Thomas B. Jacobs, and Sven Eyckerman. 2024. “Leveraging a Self-Cleaving Peptide for Tailored Control in Proximity Labeling Proteomics.” CELL REPORTS METHODS 4 (7): 100818. doi:10.1016/j.crmeth.2024.100818.
Vancouver
1.
Delhaye L, Moschonas G, Fijalkowska D, Verhee A, De Sutter D, Van de Steene T, et al. Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics. CELL REPORTS METHODS. 2024;4(7):100818.
IEEE
[1]
L. Delhaye et al., “Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics,” CELL REPORTS METHODS, vol. 4, no. 7, p. 100818, 2024.
@article{01J3N2A2P5S3292HE9723WD8ZQ,
  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 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.}},
  articleno    = {{100818}},
  author       = {{Delhaye, Louis and Moschonas, Georgios and Fijalkowska, Daria and Verhee, Annick and De Sutter, Delphine and Van de Steene, Tessa and De Meyer, Margaux and Grzesik, Hanna and Van Moortel, Laura and De Bosscher, Karolien and Jacobs, Thomas B. and Eyckerman, Sven}},
  issn         = {{2667-2375}},
  journal      = {{CELL REPORTS METHODS}},
  keywords     = {{GLUCOCORTICOID-RECEPTOR,PRECISION,MECHANISM,PROTEINS}},
  language     = {{eng}},
  number       = {{7}},
  pages        = {{21}},
  publisher    = {{Cell Press}},
  title        = {{Leveraging a self-cleaving peptide for tailored control in proximity labeling proteomics}},
  url          = {{http://doi.org/10.1016/j.crmeth.2024.100818}},
  volume       = {{4}},
  year         = {{2024}},
}
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