Proteolytic cleavage of glucagon-like peptide-1 by pancreatic beta cells and by fetal calf serum analyzed by mass spectrometry.
Fetal calf serum and a beta-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 beta cells depending on GLP-1 stimulation, the insulin and insulin fragments of the cell culture supernatants were also analyzed by this method.
Tammen
H
H
The Lower Saxony Institute for Peptide Research (IPF), Hannover, Germany.
Forssmann
W G
WG
Richter
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001769:Blood; D002417:Cattle; D005934:Glucagon / Q000737:chemistry / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1; D006868:Hydrolysis; D007515:Islets of Langerhans / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D011498:Protein Precursors; D052216:Glucagon-Like Peptide 1; D005934:Glucagon