Multifaceted challenges with liquid chromatography mass spectrometry determination of bioactive hormones secreted from stem cell-derived islet organoids

Abstract

Understanding islet cell biology is important for multiple metabolic diseases, including diabetes and obesity. Stem cell-derived islet organoids (SC-islets) are cell models suitable for closing existing knowledge gaps concerning glucose regulatory hormones, such as insulin, somatostatin-14, glucagon, and urocortin-3. The study aimed to obtain a highly selective, sensitive, versatile, and reliable determination of hormone secretion from SC-islets using liquid chromatography (LC) coupled with mass spectrometry (MS). The dissertation comments on the following research question: How far can we push LC separation and MS detection of native peptides secreted from SC-islets, and what are the limiting factors? The dissertation found that successful determination could be obtained by tailoring the LC-MS platform toward the desired analyte and sample matrices. The limiting factors concerning LC separation of bioactive peptides were; 1) non-defined adsorption depending on column format, tubing material, and sample matrices, 2) charge state distribution of multiply charged peptides combated with MP composition, and 3) compatibility towards biologically relevant sample matrices: Krebs buffer and cell medium, requiring rugged columns and instrumental set-up. The main factor to consider when applying MS for determination of bioactive peptides was the presence of disulfide bonds, which required different sample preparation depending on the applied MS instrument.