Isolation of oligopeptides from the water-soluble extract of goat cheese and their identification by mass spectrometry

A procedure for the separation and identification of small peptides from th e water-soluble fraction of a goat cheese was developed. The water-soluble extract was ultrafiltered (1000 Da membrane cutoff), and peptides were isol ated by sequential chromatography: size exclusion chromatography (HPLC-grad e water), anion exchange chromatography (phosphate buffer gradient), and se mipreparative reverse-phase high-performance liquid chromatography (water/a cetonitrile gradient). The fractions obtained were analyzed by combined mas s spectrometry methods including electrospray ionization, liquid secondary ionization, and tandem mass spectrometry to identify and to confirm the seq uences of 28 tri- to octapeptides naturally appearing in goat cheese during ripening. Among these peptides, 26 are produced by degradation of caseins but do not correspond to the known specific cleavages due to chymosin. Only low correlation was found between hydrophobicity of peptides and HPLC elut ion time with acetonitrile gradient.