Optimized LC-MS/MS Method for the Detection of ppCCK(21-44): A Surrogate to Monitor Human Cholecystokinin Secretion.

The hormone cholecystokinin (CCK) is secreted postprandially from duodenal enteroendocrine cells and circulates in the low picomolar range. Detection of this digestion and appetite-regulating hormone currently relies on the use of immunoassays, many of which suffer from insufficient sensitivity in the physiological range and cross-reactivity problems with gastrin, which circulates at higher plasma concentrations. As an alternative to existing techniques, a liquid chromatography and mass spectrometry-based method was developed to measure CCK-derived peptides in cell culture supernatants. The method was initially applied to organoid studies and was capable of detecting both CCK8 and an N-terminal peptide fragment (prepro) ppCCK(21-44) in supernatants following stimulation. Extraction optimization was performed using statistical modeling software, enabling a quantitative LC-MS/MS method for ppCCK(21-44) capable of detecting this peptide in the low pM range in human plasma and secretion buffer solutions. Plasma samples from healthy individuals receiving a standardized meal (Ensure) after an overnight fast were analyzed; however, the method only had sensitivity to detect ppCCK(21-44). Secretion studies employing human intestinal organoids and meal studies in healthy volunteers confirmed that ppCCK(21-44) is a suitable surrogate analyte for measuring the release of CCK in vitro and in vivo.

Peptidomics: A Review of Clinical Applications and Methodologies.

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.

The Human and Mouse Islet Peptidome: Effects of Obesity and Type 2 Diabetes, and Assessment of Intraislet Production of Glucagon-like Peptide-1.

To characterize the impact of metabolic disease on the peptidome of human and mouse pancreatic islets, LC-MS was used to analyze extracts of human and mouse islets, purified mouse alpha, beta, and delta cells, supernatants from mouse islet incubations, and plasma from patients with type 2 diabetes. Islets were obtained from healthy and type 2 diabetic human donors, and mice on chow or high fat diet. All major islet hormones were detected in lysed islets as well as numerous peptides from vesicular proteins including granins and processing enzymes. Glucose-dependent insulinotropic peptide (GIP) was not detectable. High fat diet modestly increased islet content of proinsulin-derived peptides in mice. Human diabetic islets contained increased content of proglucagon-derived peptides at the expense of insulin, but no evident prohormone processing defects. Diabetic plasma, however, contained increased ratios of proinsulin and des-31,32-proinsulin to insulin. Active GLP-1 was detectable in human and mouse islets but 100-1000-fold less abundant than glucagon. LC-MS offers advantages over antibody-based approaches for identifying exact peptide sequences, and revealed a shift toward islet insulin production in high fat fed mice, and toward proglucagon production in type 2 diabetes, with no evidence of systematic defective prohormone processing.

Selective stimulation of colonic L cells improves metabolic outcomes in mice.

AIMS/HYPOTHESIS: Insulin-like peptide-5 (INSL5) is found only in distal colonic L cells, which co-express glucagon-like peptide-1 (GLP-1) and peptide YY (PYY). GLP-1 is a well-known insulin secretagogue, and GLP-1 and PYY are anorexigenic, whereas INSL5 is considered orexigenic. We aimed to clarify the metabolic impact of selective stimulation of distal colonic L cells in mice. METHODS: Insl5 promoter-driven expression of Gq-coupled Designer Receptor Exclusively Activated by Designer Drugs (DREADD) was employed to activate distal colonic L cells (LdistalDq). IPGTT and food intake were assessed with and without DREADD activation. RESULTS: LdistalDq cell stimulation with clozapine N-oxide (CNO; 0.3 mg/kg i.p.) increased plasma GLP-1 and PYY (2.67- and 3.31-fold, respectively); INSL5 was not measurable in plasma but was co-secreted with GLP-1 and PYY in vitro. IPGTT (2 g/kg body weight) revealed significantly improved glucose tolerance following CNO injection. CNO-treated mice also exhibited reduced food intake and body weight after 24 h, and increased defecation, the latter being sensitive to 5-hydroxytryptamine (5-HT) receptor 3 inhibition. Pre-treatment with a GLP1 receptor-blocking antibody neutralised the CNO-dependent improvement in glucose tolerance but did not affect the reduction in food intake, and an independent group of animals pair-fed to the CNO-treatment group demonstrated attenuated weight loss. Pre-treatment with JNJ-31020028, a neuropeptide Y receptor type 2 antagonist, abolished the CNO-dependent effect on food intake. Assessment of whole body physiology in metabolic cages revealed LdistalDq cell stimulation increased energy expenditure and increased activity. Acute CNO-induced food intake and glucose homeostasis outcomes were maintained after 2 weeks on a high-fat diet. CONCLUSIONS/INTERPRETATION: This proof-of-concept study demonstrates that selective distal colonic L cell stimulation has beneficial metabolic outcomes. Graphical abstract.

Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients.

RATIONALE: Meal ingestion triggers secretion of a variety of gut and endocrine peptides important in diabetes research which are routinely measured by immunoassays. However, similarities between some peptides (glucagon, oxyntomodulin and glicentin) can cause specificity issues with immunoassays. We used a liquid chromatography/tandem mass spectrometry (LC/MS/MS) methodology to unambiguously monitor multiple gut peptides in human plasma. METHODS: A simple acetonitrile-based protein precipitation step, followed by evaporation and solid-phase extraction, removed high-abundance proteins from samples prior to nano-LC/MS/MS analysis on an Orbitrap Q-Exactive Plus mass spectrometer using a data-dependent methodology. Database searching using PEAKS identified multiple gut-derived peptides, including peptides in the mid-pg/mL range. The relative levels of these and previously characterised peptides were assessed in plasma samples from gastrectomised and control subjects during an oral glucose tolerance test. RESULTS: Analysis of plasma extracts revealed significantly elevated levels of a number of peptides following glucose ingestion in subjects who had undergone gastrectomy compared with controls. These included GLP-1(7-36), GLP-1(9-36), glicentin, oxyntomodulin, GIP(1-42), GIP(3-42), PYY(1-36), PYY(3-36), neurotensin, insulin and C-peptide. Motilin levels decreased following glucose ingestion. Results showed good correlation with immunoassay-derived concentrations of some peptides in the same samples. The gastrectomy group also had higher, but non-glucose-dependent, circulating levels of peptides from PIGR and DMBT1. CONCLUSIONS: Overall, the approach showed that a fast, generic and reproducible LC/MS/MS methodology requiring only a small volume of plasma was capable of the multiplexed detection of a variety of diabetes-related peptides.

Urotensin-II peptidomimetic incorporating a non-reducible 1,5-triazole disulfide bond reveals a pseudo-irreversible covalent binding mechanism to the urotensin G-protein coupled receptor.

The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a ß-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5-Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4-11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [125I]UII binding and [Ca2+]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+]i. Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR.

Motilin fluctuations in healthy volunteers determined by liquid chromatography mass spectrometry.

Introduction: Motilin is a hormone secreted by specialised enteroendocrine cells in the small intestine, and is known to modulate gastrointestinal motility in humans, regulating the migratory motor complex. It is understudied at least in part due to the lack of commercially available immunoassays. Method: A multiplexed liquid chromatography mass spectrometry (LC-MS/MS) method was optimised to measure motilin, insulin, C-peptide, GIP (1-42) and GIP (3-42). Corresponding active ghrelin concentrations were determined by immunoassay. Ten healthy volunteers with no prior history of gastroenterological or endocrine condition attended after overnight fast and had blood samples taken every 15 minutes for 4 hours whilst continuing to fast, and then further sampling for 2 hours following a liquid mixed meal. Hunger scores were taken at each time point using a visual analogue scale. Normal bowel habit was confirmed by 1 week stool diary. Results: Motilin levels fluctuated in the fasting state with an average period between peaks of 109.5 mins (SD:30.0), but with no evidence of a relationship with either ghrelin levels or hunger scores. The mixed meal interrupted cyclical motilin fluctuations, increased concentrations of motilin, insulin, C-peptide, GIP(1-42) and GIP(3-42), and suppressed ghrelin levels. Discussion: This study highlights the utility of LC-MS/MS for parallel measurement of motilin alongside other peptide hormones, and supports previous reports of the cyclical nature of motilin levels in the fasting state and interruption with feeding. This analytical method has utility for further clinical studies into motilin and gut hormone physiology in human volunteers.

Optimized LC-MS/MS methods for quantifying antibody-drug conjugate payloads in cell culture media containing phenol red.

Aim: Phenol red is commonly used in cell culture media, but can be detrimental to bioanalysis of in vitro samples as it may impact instrument reliability. Many researchers do their final stage of culture in 'phenol red free' media, but in collaborative work this is not always feasible.Materials & methods: A comparison was made between typical extraction methods to reduce phenol red matrix interferences, including organic solvent precipitation and solid phase extraction.Results: The final method was demonstrated to be precise and accurate for the measurement of a target analyte by LC-MS/MS, and was applied to an in vitro ADC deconjugation study.Conclusion: This method allows for for continued bioanalytical support of in vitro models used in drug development.

[Box: see text].

Post-disaster Gulf Coast recovery using telehealth.

OBJECTIVE: The Gulf Coast continues to struggle with service need far outpacing available resources. Since 2005, the Regional Coordinating Center for Hurricane Response (RCC) at Morehouse School of Medicine, Atlanta, GA, has supported telehealth solutions designed to meet high service needs (e.g., psychiatry) within primary care and other healthcare organizations. The overall RCC vision is to support autonomous, useful, and sustainable telehealth programs towards mitigating unmet disaster-related needs. SUBJECTS AND METHODS: To assess Gulf Coast telehealth experiences, we conducted semistructured interviews with both regional key informants and national organizations with Gulf Coast recovery interests. Using qualitative-descriptive analysis, interview transcripts were analyzed to identify shared development themes. RESULTS: Thirty-eight key informants were interviewed, representing a 77.6% participation rate among organizations engaged by the RCC. Seven elements critical to telehealth success were identified: Funding, Regulatory, Workflow, Attitudes, Personnel, Technology, and Evaluation. These key informant accounts reveal shared insights with telehealth regarding successes, challenges, and recommendations. CONCLUSIONS: The seven elements critical to telehealth success both confirm and organize development principles from a diverse collective of healthcare stakeholders. The structured nature of these insights suggests a generalizable framework upon which other organizations might develop telehealth strategies toward addressing high service needs with limited resources.

Rapid and Quantitative Enrichment of Peptides from Plasma for Mass Spectrometric Analysis.

Mass spectrometric analysis of peptides enables the assignment of their exact mass and confirmation of all or a significant portion of the peptide's amino acid sequence. LC-MS/MS analysis has proven invaluable in peptidomics research and can identify new biomarkers and assign their circulatory concentrations to aid research into disease processes. However, due to the high background plasma protein content, which masks the presence of the naturally low abundance circulatory peptidome, extraction of peptides from plasma prior to mass spectrometric analysis is therefore crucial. Organic solvents efficiently precipitate these high molecular weight plasma proteins while leaving small molecular weight peptides in solution, providing a rapid and effective technique for separating peptides from the contaminating plasma proteins. A secondary cleanup step involving solid phase extraction is required to remove lipids and highly hydrophobic contaminants before LC-MS/MS analysis. The method described within this chapter is effective at enriching circulatory plasma peptides prior to LC-MS/MS analysis and has been used in multiple peptidomic studies to improve peptide detection and quantification. Peptides studied using this methodology include insulin, C-peptide, glucagon, PYY, GIP, and a number of other challenging gut peptide hormones. Quantitative analyses of peptides using the described method showed good correlation with existing immunoassays.

LC-MS/MS based detection of circulating proinsulin derived peptides in patients with altered pancreatic beta cell function.

Routine immunoassays for insulin and C-peptide have the potential to cross-react with partially processed proinsulin products, although in healthy patients these are present at such low levels that the interference is insignificant. Elevated concentrations of proinsulin and des-31,32 proinsulin arising from pathological conditions, or injected insulin analogues, however can cause significant assay interferences, complicating interpretation. Clinical diagnosis and management therefore sometimes require methods that can distinguish true insulin and C-peptide from partially processed proinsulin or injected insulin analogues. In this scenario, the high specificity of mass spectrometric analysis offers potential benefit for patient care. A high throughput targeted LC-MS/MS method was developed as a fit for purpose investigation of insulin, insulin analogues, C-peptide and proinsulin processing intermediates in plasma samples from different patient groups. Using calibration standards and bovine insulin as an internal standard, absolute concentrations of insulin and C-peptide were quantified across a nominal human plasma postprandial range and correlated strongly with immunoassay-based measurements. The ability to distinguish between insulin, insulin analogues and proinsulin intermediates in a single extraction is an improvement over existing immunological based techniques, offering the advantage of exact identification of the species being measured. The method promises to aid in the detection of circulating peptides which have previously been overlooked but may interfere with standard insulin and C-peptide immunoassays.

Stimulation of motilin secretion by bile, free fatty acids, and acidification in human duodenal organoids.

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.

Organoid Sample Preparation and Extraction for LC-MS Peptidomics.

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).

Labeling and Characterization of Human GLP-1-Secreting L-cells in Primary Ileal Organoid Culture.

Glucagon-like peptide-1 (GLP-1) from intestinal L-cells stimulates insulin secretion and reduces appetite after food ingestion, and it is the basis for drugs against type-2 diabetes and obesity. Drugs targeting L- and other enteroendocrine cells are under development, with the aim to mimic endocrine effects of gastric bypass surgery, but they are difficult to develop without human L-cell models. Human ileal organoids, engineered by CRISPR-Cas9, express the fluorescent protein Venus in the proglucagon locus, enabling maintenance of live, identifiable human L-cells in culture. Fluorescence-activated cell sorting (FACS)-purified organoid-derived L-cells, analyzed by RNA sequencing (RNA-seq), express hormones, receptors, and ion channels, largely typical of their murine counterparts. L-cells are electrically active and exhibit membrane depolarization and calcium elevations in response to G-protein-coupled receptor ligands. Organoids secrete hormones in response to glucose and other stimuli. The ability to label and maintain human L-cells in organoid culture opens avenues to explore L-cell function and develop drugs targeting the human enteroendocrine system.

Insuring continuity of care for chronic disease patients after a disaster: key preparedness elements.

BACKGROUND: Care for patients with chronic diseases is a challenge after a disaster. This is particularly true for individuals from health disparate populations as they are less likely to evacuate, have fewer financial resources and often depend on resource-strapped institutions for their care. The specific aim of the study presented here was to elicit challenges and solutions in the provision of health care to those with chronic diseases after Hurricane Katrina in coastal Alabama and Mississippi. METHODS: Focusing on agencies providing care to health disparate populations, a qualitative methodology was employed using in-depth interviews with health and social service providers. Participants identified key elements essential to disaster preparedness. RESULTS: Predisaster issues were patient education and preparedness, evacuation, special needs shelters, and health care provider preparedness. Postdisaster issues were communication, volunteer coordination, and donation management. CONCLUSIONS: Lessons learned from those on the ground administering health care during disasters should inform future disaster preparations. Furthermore, the methodological approach used in this study engendered collaboration between health care institutions and may enhance future interagency disaster preparedness.

Providing continuity of care for chronic diseases in the aftermath of Katrina: from field experience to policy recommendations.

This study sought to elicit challenges and solutions in the provision of health care to those with chronic diseases after Hurricane Katrina in coastal Alabama and Mississippi. In-depth interviews with 30 health and social service providers (key informants) and 4 focus groups with patients with chronic diseases were conducted. Subsequently an advisory panel of key informants was convened. Findings were summarized and key informants submitted additional feedback. The chronic diseases identified as medical management priorities by key informants were mental health, diabetes mellitus, hypertension, respiratory illness, end-stage renal disease, cardiovascular disease, and cancer. The most frequently mentioned barrier to providing care was maintaining continuity of medications. Contributing factors were inadequate information (inaccessible medical records, poor patient knowledge) and financial constraints. Implemented or suggested solutions included relaxation of insurance limitations preventing advance prescription refills; better predisaster patient education to improve medical knowledge; promotion of personal health records; support for information technology systems at community health centers, in particular electronic medical records; improved allocation of donated medications/medical supplies (centralized coordination, decentralized distribution); and networking between local responders and external aid.