Combination of enzymatic and mass fragmentographic assays for the identification and measurement of [met5]-enkephalin

Abstract

Dipeptidyl-aminopeptidase I (DAP I) was used to hydrolyze the pentapeptide-[met5]-enkephalin into the dipeptides Tyr-Gly and Gly-Phe and methionine. The dipeptides could be derivatized and resolved by gas chromagraphy (GC); quantification of these dipeptides was obtained by single ion monitoring. Tissue samples were prepurified with Bio-Beads SM-2 and used as substrate for DAP I. The yield of methionine and of the two dipeptides increases with time. Since the same dipeptides are produced by [met5]-enkephalin (ME) and [leu5]-enkephalin, the use of the ratios of the quantities of the two dipeptides with methionine could be used for recognition. Since the only internal standard available was one with deuterated methionine, the measurement of methionine released by DAP I from the prepurified tissue could be used to measure ME content in tissues that contain a small amount of heterogeneity in the molecular forms of ME. The present GC-mass spectrometric method can be used to quantify ME in brain but not in adrenal medulla because this tissue contains a high degree of molecular heterogeneity.