title: Simultaneous quantification of intracellular and secreted active and inactive glucagon-like peptide-1 from cultured cells.
abstract: Glucagon-like peptide-1 (GLP-1) is an incretin peptide that regulates islet hormone secretion. During recent years, incretin-based therapies have been widely used for patients with type 2 diabetes. GLP-1 peptides undergo N- and C-terminal processing for gain or loss of functions. We developed a method to quantify picomolar quantities of intact GLP-1 peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By employing this label-free selected reaction monitoring (SRM) method, we were able to analyze secreted GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amid from human enteroendocrine NCI-H716 cells after stimulation with nateglinide, glucose, and sucralose. The absolute total concentrations of secreted GLP-1 peptides at baseline and after stimulation with nateglinide, glucose, and sucralose were 167.3, 498.9, 238.3, and 143.1 pM, respectively. Meanwhile, the ratios of GLP-1(1-37), GLP-1(7-37), and GLP-1(7-36 amide) to total GLP-1 peptides were similar (6 ± 3, 26 ± 3, and 78 ± 5%, respectively). The SRM assay can analyze the concentrations of individual GLP-1 peptides and, therefore, is a tool to investigate the physiological roles of GLP-1 peptides. Furthermore, the molecular species secreted from NCI-H716 cells were unknown. Therefore, we performed a secretopeptidome analysis of supernatants collected from cultured NCI-H716 cells. Together with GLP-1 peptides, we detected neuroendocrine convertase 1, which regulates peptide hormones released from intestinal endocrine L-cells.
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan.
authors.forename: Michiko
authors.initials: M
authors.lastname: Amao
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan.
authors.forename: Yoshiro
authors.initials: Y
authors.lastname: Kitahara
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Tokunaga
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan. Electronic address: kazutaka_shinbo@ajinomoto.com.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Shimbo
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan.
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Eto
authors.affiliation: Institute for Innovation, Ajinomoto, Kawasaki 210-8681, Japan.
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Yamada
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid; D052216:Glucagon-Like Peptide 1 / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D040901:Proteomics / Q000379:methods*
chemical_list: D052216:Glucagon-Like Peptide 1
keywords: Absolute quantification; Glucagon-like peptide-1; Incretin; LC–MS/MS; Secretopeptidome; Selected reaction monitoring