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Organoid Sample Preparation and Extraction for LC-MS Peptidomics.

STAR protocols (2020-12-31)
Emily L Miedzybrodzka, Rachel E Foreman, Sam G Galvin, Pierre Larraufie, Amy L George, Deborah A Goldspink, Frank Reimann, Fiona M Gribble, Richard G Kay
ABSTRACT

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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin solution, 10% in DPBS, low endotoxin, fatty acid free, suitable for cell culture, sterile-filtered
Sigma-Aldrich
3-Isobutyl-1-methylxanthine, BioUltra, ≥99%
Sigma-Aldrich
DL-Dithiothreitol, ≥99.0% (RT)
Sigma-Aldrich
D-(+)-Glucose, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.5%
Millipore
ReBlot Plus Kit
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Hanks′ Balanced Salt solution, Modified, with sodium bicarbonate, without calcium chloride and magnesium sulfate, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
Forskolin, from Coleus forskohlii, ≥98% (HPLC), powder
Sigma-Aldrich
Iodoacetamide, BioUltra