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A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids

Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was...

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Autores principales: Gonneaud, Alexis, Jones, Christine, Turgeon, Naomie, Lévesque, Dominique, Asselin, Claude, Boudreau, François, Boisvert, François-Michel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128881/
https://www.ncbi.nlm.nih.gov/pubmed/27901089
http://dx.doi.org/10.1038/srep38195
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author Gonneaud, Alexis
Jones, Christine
Turgeon, Naomie
Lévesque, Dominique
Asselin, Claude
Boudreau, François
Boisvert, François-Michel
author_facet Gonneaud, Alexis
Jones, Christine
Turgeon, Naomie
Lévesque, Dominique
Asselin, Claude
Boudreau, François
Boisvert, François-Michel
author_sort Gonneaud, Alexis
collection PubMed
description Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was applied to many other organs, including prostate, liver, kidney and pancreas. We describe here how to apply a SILAC-based quantitative proteomic approach to measure protein expression changes in intestinal organoids under different experimental conditions. We generated SILAC organoid media that allow organoids to grow and differentiate normally, and confirmed the incorporation of isotopically labelled amino acids. Furthermore, we used a treatment reported to affect organoid differentiation to demonstrate the reproducibility of the quantification using this approach and to validate the identification of proteins that correlate with the inhibition of cellular growth and development. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated this approach and showed that large-scale proteome variations can be measured in an “organ-like” system.
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spelling pubmed-51288812016-12-15 A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids Gonneaud, Alexis Jones, Christine Turgeon, Naomie Lévesque, Dominique Asselin, Claude Boudreau, François Boisvert, François-Michel Sci Rep Article Organoids have the potential to bridge 3D cell culture to tissue physiology by providing a model resembling in vivo organs. Long-term growing organoids were first isolated from intestinal crypt cells and recreated the renewing intestinal epithelial niche. Since then, this technical breakthrough was applied to many other organs, including prostate, liver, kidney and pancreas. We describe here how to apply a SILAC-based quantitative proteomic approach to measure protein expression changes in intestinal organoids under different experimental conditions. We generated SILAC organoid media that allow organoids to grow and differentiate normally, and confirmed the incorporation of isotopically labelled amino acids. Furthermore, we used a treatment reported to affect organoid differentiation to demonstrate the reproducibility of the quantification using this approach and to validate the identification of proteins that correlate with the inhibition of cellular growth and development. With the combined use of quantitative mass spectrometry, SILAC and organoid culture, we validated this approach and showed that large-scale proteome variations can be measured in an “organ-like” system. Nature Publishing Group 2016-11-30 /pmc/articles/PMC5128881/ /pubmed/27901089 http://dx.doi.org/10.1038/srep38195 Text en Copyright © 2016, The Author(s) http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Gonneaud, Alexis
Jones, Christine
Turgeon, Naomie
Lévesque, Dominique
Asselin, Claude
Boudreau, François
Boisvert, François-Michel
A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title_full A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title_fullStr A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title_full_unstemmed A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title_short A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids
title_sort silac-based method for quantitative proteomic analysis of intestinal organoids
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128881/
https://www.ncbi.nlm.nih.gov/pubmed/27901089
http://dx.doi.org/10.1038/srep38195
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