title: Chromogranin A from bovine adrenal medulla: molecular characterization of glycosylations, phosphorylations, and sequence heterogeneities by mass spectrometry.
abstract: Chromogranin A (CGA) is a member of a family of acidic glycoproteins present in endocrine and neuroendocrine tissues. One of its suggested physiological roles is being a precursor molecule for several peptide hormons. Further interest in this protein has recently originated from its potential role in pathophysiological processes of Alzheimer's disease. The concentration of CGA in the brain has been used for diagnosis of this disease, and CGA as an insoluble deposit has been found in the extracellular beta-amyloid plaques. By developing a new purification procedure we were able to isolate abundant CGA in high purity from bovine chromaffin cells. A MALDI-MS analysis of the intact protein revealed a heterogeneous molecular mass of ca. 50 kDa, indicating several structure modifications. By use of several subsequent proteolytic/chemical cleavage steps, HPLC isolation, a newly developed deglycosylation procedure, and several MS and MS-MS fragmentation approaches, the complete primary structure of CGA including four sequence heterogeneities, two O-glycosylations, five phosphorylations, and one disulfide bridge could be characterized. For both glycans six different forms could be identified. Ser167 was found to be mainly glycosylated by a trisaccharide, and Thr231 was found to be mainly glycosylated by a tetrasaccharide. Ser81, Ser124, and Ser297 residues were partially phosphorylated, whereas Ser372 and Ser377 were found completely phosphorylated. Sequence heterogeneities were identified in positions 293 (H/R), 301 (K/E), and 373 (Q/R) and at the partly missing C-terminal residue. Furthermore, a disulfide bridge between Cys17 and Cys38 was ascertained.
authors.affiliation: Department of Chemistry, University of Konstanz, Konstanz, D-78457, Germany.
authors.forename: S H
authors.initials: SH
authors.lastname: Bauer
authors.forename: X Y
authors.initials: XY
authors.lastname: Zhang
authors.forename: W
authors.initials: W
authors.lastname: Van Dongen
authors.forename: M
authors.initials: M
authors.lastname: Claeys
authors.forename: M
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000313:Adrenal Medulla / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002837:Chromaffin Granules / Q000737:chemistry*; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004220:Disulfides / Q000032:analysis; D006031:Glycosylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods; D014357:Trypsin
chemical_list: D053379:Chromogranin A; D002864:Chromogranins; D004220:Disulfides; D010446:Peptide Fragments; D014357:Trypsin