Application of high-performance liquid chromatography in neurophysin disulfide assignment

The combined use of ion-exchange, and reversed-phase high-performance liquid chromatography (HPLC) for the isolation of cystine-containing peptides from highly heterogeneous products of the proteolytic digestion of bovine neurophysins is described. The protein was sequentially cleaved by enzymes of decreasing specificity; the peptides released were initially fractionated by gel chromatography and then purified by HPLC. The purified peptides were analyzed by determination of their amino acid composition and mass spectrometry, supported by sequencing techniques. Three of the seven disulfide pairs of neurophysin have now been assigned. The usefulness of the combined use of HPLC and mass spectrometry in assigning these and the other disulfide pairs is illustrated.