SEQUENCE-ANALYSIS OF CYSTEINE AND CYSTINE CONTAINING PEPTIDES AND PROTEINS BY A GAS-PHASE SEQUENCER WITH ISOCRATIC SEPARATION OF PTH-AMINO ACIDS

A facile method for routine sequence analysis of cysteine and cystine containing peptides or proteins is described. An automated gas-phase protein sequencer, Shimadzu PSQ-1 System, consisting of an Edman-reaction unit and an isocratic separation mode for the phenylthiohydantoin amino acid analysis with equivalent sensitivity and resolution to that of the current gradient mode, allows easy determination of the intact second half cystine, of which phenylthiohydantoin derivatives appear as a dithiothreitol adduct of dehydroalanine, cysteine and S-pyridylethylated cysteine. The optimal sequencing protocol for in situ pyridylethylation was established. This protocol gives an N-terminal amino acid sequence with half cystines, which are automatically converted to the S-pyridylethyl derivative in the sequencer. Sequence analyses both intact and S-pyridylethylated cysteine can identify a disulfide form if only one disulfide linkage exists in the sample. Sequence analysis of synthetic peptides containing various S-protected cysteines is also described. This analysis allows examination of the correct chain assembly before disulfide bond formation.