Isolation and mass spectrometric characterization of an oxidized form of vasostatin I, an N-terminal chromogranin A-derived protein, from bovine chromaffin cells
✍ Scribed by L. Dillen; X. Y. Zhang; M. Claeys; F. Liang; W. P. De Potter; W. Van Dongen; E. L. Esmans
- Publisher
- John Wiley and Sons
- Year
- 1995
- Tongue
- English
- Weight
- 538 KB
- Volume
- 30
- Category
- Article
- ISSN
- 1076-5174
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
An N‐terminal proteolytic processing product of chromogranin A was obtained from bovine chromaffin granules using two steps of C~18~ 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 (CGA~1−76~). Derivatization with mercaptoethanol showed that the peptide had an intact SS 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 C~18~ 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.