Post-translational processing of preprosomatostatin-II examined using fast atom bombardment mass spectrometry.
The products and an intermediate of preprosomatostatin-II processing in the anglerfish islet were purified and subjected to structural analysis. The peptides isolated identify the site of signal cleavage (between Ser-24 and Gln-25). The prohormone is further processed at Arg-97 and, to a lesser extent, at the two adjacent basic amino acid residues Lys-61 and Arg-62. A 28-residue somatostatin is also generated which can be hydroxylated at Lys-23. A proteolytic processing site which would form the 14-residue somatostatin does not appear to be used to a significant degree. Fast atom bombardment mass spectrometry (FABMS) was used to demonstrate that the amino-terminal residues of peptides 25-60, and 25-90 are pyroglutamic acid, a modification which precludes Edman degradation of these peptides. Analysis of the peptides and tryptic peptide maps by FABMS allowed confirmation of the sites of prohormone conversion and indicated that terminal basic residues were removed during processing. Three amino acid residues were also found to differ from the amino acid sequence deduced from the cDNA and were localized to specific regions by FABMS analysis. Residues found to differ from the cDNA (cDNA in parentheses) were: Asp-77 (Thr), Val-78 (Phe), and Gly-90 (Glu). Mass assignments were confirmed by running a single cycle of Edman degradation prior to FABMS. The peptides noted above were also examined by Edman sequence analysis. The sequence of a cDNA clone to preprosomatostatin-II was re-examined in light of the observed differences at the protein level. This study emphasizes the utility of FABMS in prohormone processing studies and in identification of post-translational processing events.
D000595:Amino Acid Sequence; D000818:Animals; D005399:Fishes / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013004:Somatostatin / Q000378:metabolism*
D010446:Peptide Fragments; D011498:Protein Precursors; D013004:Somatostatin