Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways.

Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models. Key features • 3D organotypic models with genetically engineered human cells. • In-depth quantitative proteomics and phosphoproteomics in 2D cell culture. • Careful handling of cell cultures is critical for the successful formation of the organotypic cultures. • For complete details on the use of this protocol, please refer to Ye et al. 2022.