The application of chemical ionization mass spectrometry to the identification of urinary peptides

Abstract

The use of chemical ionization (c.i.) mass spectrometry has been applied to the identification of complex mixtures of dipeptides, without the need for a prior separation of the components. The peptides are identified as their N-ethoxycarbonylpropenyl dimethyltrideuteromethyl ester derivatives. The technique was originally used by us for the identification of dipeptide mixtures from dipeptidylaminopeptidase-I digests of polypeptides, but was found to be a valuable procedure for the rapid identification of urinary peptides. The protocol of the procedure involves: (1) purification of the urinary peptides by column chromatography on G10 Sephadex followed by paper chromatography; (2) derivatization of the peptide fraction with ethylacetoacetate and dimethyltrideuteromethyl-phenylammonium hydroxide; (3) mass spectrometry (c.i.) of the derivatized fraction; (4) identification of the urinary dipeptides by computer matching of selected ions from the mass spectrum. This procedure was applied to the identification of dipeptides in the urine of a child suffering from chronic skin ulceration and oedema. At least 15 dipeptides were identified, most of which contained proline or hydroxyproline in the carboxyl terminal position. The results suggested that the patient suffered from a defect in collagen metabolism. This hypothesis was subsequently confirmed by a grossly diminished level of prolidase in cultured fibroblasts.