Determination of disulfide bridges in natural and recombinant insect defensin A

Abstract

The primary-structure comparison of natural insect defensin A from Phormia terranovae and recombinant insect defensin A from Saccharomyces cerevisiae has been accomplished using a combination of Edman degradation and liquid secondary ion mass spectrometry. The natural and recombinant proteins have the same primary structure with identical disulfide-bond designations (formula; see text) as determined from the peptides obtained after thermolysin digestion. The combined use of Edman degradation and mass spectometry allowed the disulfide-bridge structure to be determined with a total of only 40 micrograms (9.9 nmol) natural peptide. Mass spectrometry provides a rapid means of disulfide-bridge verification, requiring not more than 20 micrograms recombinant insect defensin A, which is compatible with use in batch analysis.