title: A SILAC-Based Method for Quantitative Proteomic Analysis of Intestinal Organoids.
abstract: 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.
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Alexis
authors.initials: A
authors.lastname: Gonneaud
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Christine
authors.initials: C
authors.lastname: Jones
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Naomie
authors.initials: N
authors.lastname: Turgeon
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Dominique
authors.initials: D
authors.lastname: Lévesque
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: Claude
authors.initials: C
authors.lastname: Asselin
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Boudreau
authors.affiliation: Department of Anatomy and Cell Biology, Université de Sherbrooke, 3201 Jean-Mignault, Sherbrooke, Québec, J1E 4K8, Canada.
authors.forename: François-Michel
authors.initials: FM
authors.lastname: Boisvert
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004847:Epithelial Cells / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D009940:Organoids / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D020543:Proteome