Proteolytic cleavage of glucagon-like peptide-1 by pancreatic β cells and by fetal calf serum analyzed by mass spectrometry

Abstract

Fetal calf serum and a β-cell line exhibit a proteolytic activity essential for the biological function of glucagon-like peptide-1 (GLP-1). This process of cleavage was investigated using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To generate processing products, GLP-1 was subjected to rat insulinoma m5F (RINm5F) cell cultures or to fetal calf serum (FCS). For detection of processing products, a standardized extraction method including ion-exchange batch extraction, ultrafiltration, gel filtration, and reversed-phase chromatography was used. The RP fractions were analyzed by MALDI-TOF-MS. Processed proteolytic products were detected by comparing the resulting mass spectra of cell media or FCS after 2 h incubation with GLP-1 (7–36) amide with these of 2 h controls. To perform the comparison of the resulting mass spectra, software (massspecanalyst) based on Microcal Software, Origins C-like language labtalk was developed. GLP-1 fragments were purified by RP-HPLC, and characterized by sequence analysis. As insulin is the major secretory product of β cells depending on GLP-1 stimulation, the insulin and insulin fragments of the cell culture supernatants were also analyzed by this method.