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Improvements in both liquid chromatography (LC) and mass spectrometry (MS) instrumentation have greatly enhanced proteomic and small molecule metabolomic analysis in recent years. Less focus has been on the improved capability to detect and quantify small bioactive peptides, even though the exact sequences of the peptide species produced can have important biological consequences. Endogenous bioactive peptide hormones, for example, are generated by the targeted and regulated cleavage of peptides from their prohormone sequence. This process may include organ specific variants, as proglucagon is converted to glucagon in the pancreas but glucagon-like peptide-1 (GLP-1) in the small intestine, with glucagon raising, whereas GLP-1, as an incretin, lowering blood glucose. Therefore, peptidomics workflows must preserve the structure of the processed peptide products to prevent the misidentification of ambiguous peptide species. The poor in vivo and in vitro stability of peptides in biological matrices is a major factor that needs to be considered when developing methods to study them. The bioinformatic analysis of peptidomics data sets requires the inclusion of specific post-translational modifications, which are critical for the function of many bioactive peptides. This review aims to discuss and contrast the various extraction, analytical, and bioinformatics approaches used for human peptidomics studies in a multitude of matrices.

Objective Motilin is a proximal small intestinal hormone with roles in gastrointestinal motility, gallbladder emptying, and hunger initiation. In vivo motilin release is stimulated by fats, bile, and duodenal acidification but the underlying molecular mechanisms of motilin secretion remain poorly understood. This study aimed to establish the key signaling pathways involved in the regulation of secretion from human motilin-expressing M-cells. Methods Human duodenal organoids were CRISPR-Cas9 modified to express the fluorescent protein Venus or the Ca2+ sensor GCaMP7s under control of the endogenous motilin promoter. This enabled the identification and purification of M-cells for bulk RNA sequencing, peptidomics, calcium imaging, and electrophysiology. Motilin secretion from 2D organoid-derived cultures was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), in parallel with other gut hormones. Results Human duodenal M-cells synthesize active forms of motilin and acyl-ghrelin in organoid culture, and also co-express cholecystokinin (CCK). Activation of the bile acid receptor GPBAR1 stimulated a 3.4-fold increase in motilin secretion and increased action potential firing. Agonists of the long-chain fatty acid receptor FFA1 and monoacylglycerol receptor GPR119 stimulated secretion by 2.4-fold and 1.5-fold, respectively. Acidification (pH 5.0) was a potent stimulus of M-cell calcium elevation and electrical activity, an effect attributable to acid-sensing ion channels, and a modest inducer of motilin release. Conclusions This study presents the first in-depth transcriptomic and functional characterization of human duodenal motilin-expressing cells. We identify several receptors important for the postprandial and interdigestive regulation of motilin release., Highlights • Mechanisms of motilin secretion were assessed in a new human duodenal organoid model. • RNA sequencing identified ion channels and receptors involved in M-cell activity. • M-cells fired action potentials similar to those in other enteroendocrine cell types. • Motilin release was triggered by low pH, bile, and free fatty acid receptor-1 ligands. • Low pH depolarized M-cells and increased calcium through acid-sensing ion channels (ASIC). Image 1

Summary 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)., Graphical Abstract, Highlights • 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.