ISOLATION AND MASS-SPECTROMETRIC CHARACTERIZATION OF AN OXIDIZED FORMOF VASOSTATIN-I, AN N-TERMINAL CHROMOGRANIN A-DERIVED PROTEIN, FROM BOVINE CHROMAFFIN CELLS

An N-terminal proteolytic processing product of chromogranin A was obt ained from bovine chromaffin granules using two steps of C-18 solid-ph ase extraction and reversed-phase high-performance liquid chromatograp hy. Electrospray mass spectrometry revealed a protein with a molecular mass of 8632.0 for the fraction showing immunoreactivity against the N-terminus of chromogranin A, which differed by 48 u from that of the N-terminal processing product, vasostatin I (CGA(1-76)). Derivatizatio n with mercaptoethanol showed that the peptide had an intact S-S bridg e, which is a key structural feature of vasostatin I. Peptide mapping experiments involving reduction/alkylation with vinylpyridine and tryp sin digestion were consistent with oxidation of the three methionine r esidues of vasostatin I to their sulphoxide forms. The oxidation of th e methionine residues was found to occur during the C-18 solid-phase e xtraction procedure, The use of freshly prepared Tris-HCl buffer and e luents and flushing the buffer and eluents with nitrogen were shown to result in the isolation of the non-oxidized form of vasostatin I.