Rapid Characterization of Natural and Biotechnologically Synthesized Human Growth Hormones by Fast Atom Bombardment Mass Spectrometry and High-Performance Liquid Chromatography

概要

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Fast atom bombardment mass spectrometric (FAB-MS) analysis of tryptic digest of human growth hormone (hGH) was caried out to verify the primary structures of two kinds of hGHs expressed in Escherichia coli; native type (r-hGH) and N-terminal methionine-containing type of hGH (met-hGH. Both structures were confirmed by comparing the mass spectra of the tryptic digest mixtures with that of authentic hGH extracted from human pituitary (p-hGH). The signals detected in the mass spectura accounted for 82% of the sequence of hGH. In order to determine the location of disulfide bonds, a method was designd for selective preparation of peptide fragments containing a disulfide bond from the tryptic digest. The selection was performed by comparison with high-performance liquid chromatograms before and after the reduction of the digest mixtures with dithiothreitol (DTT). From the mass spectra of two disulfide-containing peptide fragments, the locations of two disulfide bonds in the hGH sequence were confirmed to be the same among the three types of hGHs. The present study indicates that the FAB-MS provides an excellent method for the rapid verification of the primary structure of proteins, and the combination of high-performance liquid chromatography (HPLC) with this method facilitates and analysis of disulfide bonds.
1988-03-25