Identification and characterization of novel chromogranin B-derived peptides from porcine chromaffin granules by liquid chromatography/electrospray tandem MS

Abstract

Chromogranin B (CgB) is a regulated secretory protein that is stored in endocrine and neuroendocrine cells. It can be processed proteolytically to small peptide fragments. In the present study three proteolytic products of porcine CgB were obtained after size-exclusion, immunoaffinity, and reversed-phase chromatography, and then identified by electrospray tandem MS. One novel peptide was identified as S586±R602 and is phosphorylated at one or two serine residues. Another novel peptide H603±Q636 (HQ-34), with molecular mass 3815.56 Da, was found to be oxidized at the methionine residue. In addition, a secretolytin-like peptide fragment (KR-11), which is two amino acids shorter than the bovine secretolytin, was found. This is the first report that the C-terminal region of CgB, the homologue of human CCB, is proteolytically processed further into three small peptide fragments.

Characterization and location of post‐translational modifications on chromogranin B from bovine adrenal medullary chromaffin granules

“…Since its characterization in 1985 [10] the genomic organization of mouse CGB has been reported [39] and amino sequences for bovine [33], human [40], rat [41], mouse [42], pig [43] and frog [44] have also been reported. The alignment of the different CGB sequences from different species indicate that both the N-terminal domain (residues 1-53) and the C-terminal domain (residue 415-626) are highly homologous, while the middle portion of the protein shows remarkable sequence variation between species (Fig.…”

“…In addition, we have demonstrated that the monophosphorylated forms of CCB (CGB 564-626 ) are predominant Figure 9. Sequence alignment of CGB from several species: B, bovine [33]; H, human [39]; P, pig [42]; R, rat [40]; M, mouse [41]; F, frog [43]. Residues in bold correspond to putative modified residues and residues underlined have been experimentally identified.…”

A Sulfated, Phosphorylated 7 kDa Secreted Peptide Characterized by Direct Analysis of Cell Culture Media

“…These studies have required extensive sample preparation, peptide purification, and subsequent proteolytic mapping of chrombacin. Wang et al isolated a 17 residue N-terminal fragment of porcine chrombacin and, based on MS/MS fragmentation, proposed phosphorylation at serine residues . The possibility of tyrosine sulfation was recognized and dismissed as unlikely based on the lack of “consensus amino acids”.…”

“…Wang et al isolated a 17 residue N-terminal fragment of porcine chrombacin and, based on MS/MS fragmentation, proposed phosphorylation at serine residues. 20 The possibility of tyrosine sulfation was recognized and dismissed as unlikely based on the lack of "consensus amino acids". More recently, Gasnier et al chromatographically purified bovine chrombacin which was subsequently subjected to successive protease digestion.…”

Neuropeptides and Chromogranins

“…Various peptides generated by proteolytic cleavage of the CgB precursor protein have already been described 20,21. Now De Potter and his group have demonstrated that four novel CgB‐derived peptides recently identified in porcine adrenal medulla22 are also present in LDVs of porcine splenic nerve and that two of them are substrates for dipeptidylpeptidase. Portela‐Gomes and Stridsberg conducted a comprehensive mapping of the different neuroendocrine cell types in the human pancreas with antibodies against 16 individual peptidergic regions of CgB.…”