Identification of the C-terminal portion of a protein by comparative peptide mapping.
A method is presented for the simple identification of C-terminal fragment of proteins. The method consists of (i) C-terminal processing of a protein by carboxypeptidase and (ii) comparative peptide mapping of the intact and carboxypeptidase-excised protein after fragmentation by endoproteinase or by chemical cleavage. The peptide mapping was performed by means of high-performance reversed-phase chromatography, where the C-terminal fragment was identified as a peptide peak that was lost or decreased in the carboxypeptidase-excised protein. The C-terminal sequence of the protein could be then determined by sequential Edman degradation of the C-terminal fragment collected from the peptide mapping chromatography. The sensitivity of the method depends solely on the peptide detection and subsequent Edman degradation, currently available techniques of which require a nanomole to subnanomole quantity of protein. The present method can be coupled with conventional carboxypeptidase technology because it utilizes a protein portion remaining after carboxypeptidase digestion while released amino acids are needed in the conventional technique. The method would be particularly valuable in finding a gene probe site for a RNA message coding for the C-terminal portion of a molecule.
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin; D002268:Carboxypeptidases; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments / Q000032:analysis*; D011506:Proteins; D014357:Trypsin; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D002147:Calmodulin; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D002268:Carboxypeptidases; D014357:Trypsin