Evidence that the chromogranin B fragment 368-417 extracted from a pheochromocytoma is phosphorylated

A rabbit antiserum was raised against a synthetic peptide corresponding to residues 403 to 417 of human chromogranin B. This peptide was chosen to mat ch the potential C-terminal end of a putative proteolytic fragment of the p rotein located between dibasic doublets in positions 366-367 and in positio ns 418-419 of the precursor. A radioimmunoassay based on this antiserum was developed and used to detect the protein or a fragment thereof in a pheoch romocytoma tumor extract. One fragment was purified to homogeneity by succe ssive reverse-phase HPLC chromatographies. The N-terminal sequence establis hed by automated Edman degradation, was N-Y-P-S-L-E-L-D-K-M-A-H-G-Y-G-E-E-S -E-E-E-R corresponding to the 368-389 sequence of human chromogranin B. Tak ing into account the specificity of the antiserum used for peptide identifi cation and alignment with the precursor sequence, we deduced that the purif ied peptide was chromogranin B (368-417) and represented a new peptide gene rated by limited proteolysis of chromogranin B. Combining electrospray mass -spectrometry and enzymatic dephosphorylation. we demonstrated that this pe ptide was phosphorylated. (C) 2001 Elsevier Science Inc. All rights reserve d.