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.

Exploring ribosome composition and newly synthesized proteins through proteomics and potential biomedical applications

“…One of the advantages of this approach is that there is a minimum experimental variation as the label is incorporated at the amino acid level and thus, analysis of mass spectra is straightforward and highly precise. Although SILAC was developed and used widely in cell cultures for quantitative proteomics of both primary cells [57] and cell lines [58], recent studies have used SILAC for in vitro cell-based organoids [59] and also in vivo [60]. An organoid is derived from induced pluripotent stem cells of a specific donor and can be considered a near-physiological 3D model that closely approximates the genetic and functional characteristics of the stem cell donor.…”

“…An organoid is derived from induced pluripotent stem cells of a specific donor and can be considered a near-physiological 3D model that closely approximates the genetic and functional characteristics of the stem cell donor. Organoids derived from a variety of induced pluripotent stem cell donors can address individual human population variability, making it useful for applications in precision medicine [59,61–62]. The complete and almost complete labeling (96.2%) by SILAC in vivo was reported for mice [63] and flies [60], respectively, and partial SILAC labeling was accomplished for chickens [40,64] and newts [65].…”

Phenotypic Analysis of Organoids by Proteomics

“…Nonetheless, prolonged organoid culture is required to obtain sufficient material as well as for organoid proliferation and differentiation prior to sample preparation for mass spectrometry, minimizing the difference of the additional time for growth needed to insure sufficient SILAC organoid labeling. Organoids, either directly or following isotope incorporation, can be frozen for storage, preservation and future assays, which shortens the time of culture after the initial incorporation . Thus, culture time differences may be minimized since SILAC‐labeled frozen organoids can be used to restart organoid growth .…”

“…Organoids, either directly or following isotope incorporation, can be frozen for storage, preservation and future assays, which shortens the time of culture after the initial incorporation . Thus, culture time differences may be minimized since SILAC‐labeled frozen organoids can be used to restart organoid growth . As opposed to isobaric labeling methods, SILAC is more limited in the number of combinations available for isotope labeling because of the number of labels that can be simultaneously used.…”

“…Gonneaud et al. have established intestinal jejunal organoid growth conditions for SILAC incorporation, including the optimal time for efficient isotope incorporation, and the possibility to freeze SILAC‐labeled organoids for future use. Treatment with the class I HDAC inhibitor CI994 induced morphological changes, resulting in smaller and less differentiated organoids characterized by an apparent reduction in intestinal epithelial crypt formation.…”