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 p rotein sequencer, Shimadzu PSQ-1 System, consisting of an Edman-reacti on unit and an isocratic separation mode for the phenylthiohydantoin a mino 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 a s a dithiothreitol adduct of dehydroalanine, cysteine and S-pyridyleth ylated cysteine. The optimal sequencing protocol for in situ pyridylet hylation 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 in tact 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 de scribed. This analysis allows examination of the correct chain assembl y before disulfide bond formation.