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用于信号通路研究的磷酸化蛋白质组学分析及器官型培养

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

作者信息

Ye Zilu, Wandall Hans H, Dabelsteen Sally

机构信息

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

出版信息

Bio Protoc. 2024 Feb 20;14(4):e4941. doi: 10.21769/BioProtoc.4941.

DOI:10.21769/BioProtoc.4941
PMID:38410375
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10895376/
Abstract

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.

摘要

信号通路参与了从胚胎发育到病理状态的关键细胞功能，在组织稳态和转化中起关键作用。尽管大多数信号通路已被深入研究，但大多数研究是在小鼠模型或简单细胞培养中进行的。我们描述了在三维器官型皮肤模型中，使用CRISPR-Cas9基因工程化的人角质形成细胞（N/TERT-1）结合定量蛋白质组学和磷酸化蛋白质组学质谱，对人组织中的TGF-β信号通路进行剖析。使用人三维器官型培养物和基因工程结合定量蛋白质组学和磷酸化蛋白质组学是一种强大的工具，可在人体环境中深入了解信号通路。这些方法适用于其他基因靶点以及三维细胞和组织模型。关键特性 • 具有基因工程化人类细胞的三维器官型模型。 • 二维细胞培养中的深入定量蛋白质组学和磷酸化蛋白质组学。 • 小心处理细胞培养物对于器官型培养物的成功形成至关重要。 • 有关本方案使用的完整详细信息，请参阅Ye等人2022年的文献。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/9e1b0390b9c6/BioProtoc-14-4-4941-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/e0a18d6420e6/BioProtoc-14-4-4941-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/74a7c92be988/BioProtoc-14-4-4941-v001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/87873263340d/BioProtoc-14-4-4941-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/19b2d62f0b0c/BioProtoc-14-4-4941-v002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/a6dacdf42447/BioProtoc-14-4-4941-v003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/a9769c206512/BioProtoc-14-4-4941-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/18c168676171/BioProtoc-14-4-4941-v004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/34603afb855a/BioProtoc-14-4-4941-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/da5ea9017ef4/BioProtoc-14-4-4941-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/96ed0f75337c/BioProtoc-14-4-4941-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/9e1b0390b9c6/BioProtoc-14-4-4941-g007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/e0a18d6420e6/BioProtoc-14-4-4941-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/74a7c92be988/BioProtoc-14-4-4941-v001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/87873263340d/BioProtoc-14-4-4941-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/19b2d62f0b0c/BioProtoc-14-4-4941-v002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/a6dacdf42447/BioProtoc-14-4-4941-v003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/a9769c206512/BioProtoc-14-4-4941-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/18c168676171/BioProtoc-14-4-4941-v004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/34603afb855a/BioProtoc-14-4-4941-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/da5ea9017ef4/BioProtoc-14-4-4941-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/96ed0f75337c/BioProtoc-14-4-4941-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/c737/10895376/9e1b0390b9c6/BioProtoc-14-4-4941-g007.jpg

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Characterization of TGF-β signaling in a human organotypic skin model reveals that loss of TGF-βRII induces invasive tissue growth.在人体器官型皮肤模型中对 TGF-β 信号的表征揭示了 TGF-βRII 的缺失会诱导侵袭性组织生长。
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Global mapping of GalNAc-T isoform-specificities and O-glycosylation site-occupancy in a tissue-forming human cell line.
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