PROTEOLYSIS OF HUMAN CALCITONIN IN EXCISED BOVINE NASAL-MUCOSA - ELUCIDATION OF THE METABOLIC PATHWAY BY LIQUID SECONDARY IONIZATION MASS-SPECTROMETRY (LSIMS) AND MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY (MALDI)

Purpose. Two calcitonins, i.e. human calcitonin (hCT) and, for compari son, salmon calcitonin (sCT), were chosen as peptide models to investi gate nasal mucosal metabolism. Methods. The susceptibility of hCT and sCT to nasal mucosal enzymes was assessed by in-and-out reflection kin etics experiments in an in vitro model based on the use of freshly exc ised bovine nasal mucosa, with the mucosal surface of the mucosa facin g the peptide solution. The kinetics of CT degradation in the bulk sol ution was monitored by HPLC. Peptide sequences of the main nasal metab olites of hCT were analyzed by using both liquid secondary ionization mass spectrometry (LSIMS), following HPLC fractionation of the metabol ites, and matrix-assisted laser desorption ionization mass (MALDI) spe ctrometry. For sCT, the molecular weights of two major metabolites wer e determined by LC-MS with electrospray ionization. Results. Both CTs were readily metabolized by nasal mucosal enzymes. In the concentratio n range studied metabolic rates were higher with hCT than with sCT. Pr esence of endopeptidase activities in the nasal mucosa was crucial, cl eaving both calcitonins in the central domain of the molecules. Conclu sions. Typically, initial metabolic cleavage of hCT in nasal mucosa is due to both chymotryptic- and tryptic-like endopeptidases. The subseq uent metabolic break-down follows the sequential pattern of aminopepti dase activity. Tryptic endopeptidase activity is characteristic of nas al sCT cleavage.