ANALYTICAL STRATEGIES IN THE STRUCTURAL CHARACTERIZATION OF ELCATONIN

Elcatonin is a synthetic peptide of 32 amino acid residues, that diffe rs from natural peptide hormone (eel calcitonin) in that the 1 and 7 c ystine residues are replaced with alpha-amino suberic acid (Asu). Elca tonin is pharmacologically important, since it inhibits osteoclastic b one reserption and induces calcium uptake from body fluids, It is also used for the treatment of Page's disease and hypercalcemic conditions , Until now the structural characterization of elcatonin has been obta ined by proteolytic digestion followed by high performance liquid chro matographic (HPLC) analysis of the peptide fragments, Capillary electr ophoresis and fast-atom bombardment have also been employed, This work describes the results obtained when a liquid chromatograph? coupled t o mass spectrometer using electrospray ionization (LC/ESI-MS) was appl ied to elcatonin analysis. After digestion with trypsin, the resulting peptides were separated by HPLC with 'on-line' UV detection, and dire ctly injected into the ESI source, The molecular weights of all the fr agments were detected, and the sequences of two of them were determine d by collisionally induced dissociation in the ESI source. To confirm these 'on-line' results, the 'off-line' approach was also applied. In this case, the fragments from tryptic digestion were isolated by prepa rative HPLC, concentrated and analyzed by direct infusion into the ESI -MS system. Then, different elcatonin digests obtained using other pro teases, e.g. protease V8 and clostripain, were analyzed by direct infu sion, and these results combined with those achieved by the 'on-line' analysis allowed us to obtain the entire mapping of elcatonin. (C) 199 7 by John Wiley & Sons, Ltd.