Organoid Sample Preparation and Extraction for LC-MS Peptidomics

• Published in: STAR protocols Vol. 1; no. 3; p. 100164
• Main Authors: Miedzybrodzka, Emily L., Foreman, Rachel E., Galvin, Sam G., Larraufie, Pierre, George, Amy L., Goldspink, Deborah A., Reimann, Frank, Gribble, Fiona M., Kay, Richard G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.12.2020; Elsevier
• Subjects: Amino Acid Sequence; Cellular Biology; Chromatography, Liquid; Flow Cytometry; Humans; Life Sciences; Mass Spectrometry; Organoids; Organoids - metabolism; Peptides - chemistry; Peptides - isolation & purification; Peptides - metabolism; Proteomics; Proteomics - methods; Protocol; Tandem Mass Spectrometry; Organoids; Mass Spectrometry; Proteomics
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2020.100164

This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models. For complete details on the use and execution of this protocol, please refer to Goldspink et al. (2020). [Display omitted] •Preparing organoid cultures, secretions, and FAC-sorted cells for peptidomics•An established pipeline covering peptide extraction, LC-MS, and data analysis•Unambiguous detection of closely related peptide hormones•Semi-quantitative analysis of secreted peptides from stimulated organoid cultures This protocol describes the peptidomic analysis of organoid lysates, FACS-purified cell populations, and 2D culture secretions by liquid chromatography mass spectrometry (LC-MS). Currently, most peptides are quantified by ELISA, limiting the peptides that can be studied. However, an LC-MS-based approach allows more peptides to be monitored. Our group has previously used LC-MS for tissue peptidomics and secretion of enteroendocrine peptides from primary culture. Now, we extend the use to organoid models.