Analytical strategies in the structural characterization of elcatonin

Elcatonin is a synthetic peptide of 32 amino acid residues, that differs from natural peptide hormone (eel calcitonin) in that the 1 and 7 cystine residues are replaced with alpha-amino suberic acid (Asu). Elcatonin is pharmacologically important, since it inhibits osteoclastic bone 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 obtained by proteolytic digestion followed by high performance liquid chromatographic (HPLC) analysis of the peptide fragments. Capillary electrophoresis and fast-atom bombardment have also been employed. This work describes the results obtained when a liquid chromatograph, coupled to mass spectrometer using electrospray ionization (LC/ESI-MS) was applied to elcatonin analysis. After digestion with trypsin, the resulting peptides were separated by HPLC with 'on-line' UV detection, and directly injected into the ESI source. The molecular weights of all the fragments were detected, and the sequences of two of them were determined 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 preparative HPLC, concentrated and analyzed by direct infusion into the ESI-MS system. Then, different elcatonin digests obtained using other proteases, e.g. protease V8 and clostripain, were analyzed by direct infusion, and these results combined with those achieved by the 'on-line' analysis allowed us to obtain the entire mapping of elcatonin.