Analytical Chemistry and Spectroscopy
Wiley InterScience Backfile Collection 1832-2000
Chemistry and Pharmacology
An N-terminal proteolytic processing product of chromogranin A was obtained from bovine chromaffin granules using two steps of C18 solid-phase extraction and reversed-phase high-performance liquid chromatography. 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 (CGA1-76). Derivatization with mercaptoethanol showed that the peptide had an intact S—S bridge, which is a key structural feature of vasostatin I. Peptide mapping experiments involving reduction/alkylation with vinylpyridine and trypsin digestion were consistent with oxidation of the three methionine residues of vasostatin I to their sulphoxide forms. The oxidation of the methionine residues was found to occur during the C18 solid-phase extraction procedure. The use of freshly prepared Tris-HCl buffer and eluents and flushing the buffer and eluents with nitrogen were shown to result in the isolation of the non-oxidized form of vasostatin I.
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