Preparation of stable isotope‐incorporated peptide internal standards for field desorption mass spectrometry quantification of peptides in biologic tissue

Stable isotopes have been incorporated into two opioid pentapeptides, leucine enkephalin and methionine enkephalin, using chemical‐ and enzymatic‐catalyzed reactions. Oxygen‐18 from H 2 18 O was the stable isotope incorporated. High‐performance liquid chromatography separated the chemical reaction mixture into three fractions: hydrolyzed peptide fraction, 18 O‐incorporated enkephalin, and 18 O‐incorporated enkephalin ester. Analysis of individual isotopic species ( 18 O 2 , 18 O 16 O, 16 O 2 ) in the latter two fractions was done with field desorption mass spectrometry. Porcine esterase II was used to hydrolyse enkephalin esters and recover stable isotope. Up to 90% 18 O 2 was incorporated. Yields of 18 O 2 species for the overall procedure were for leucine enkephalin, 56%, and for methionine enkephalin, 38%. This scheme represents the first fast and facile preparation of a stable isotope‐incorporated peptide internal standard for use in measuring peptides in biologic extracts and is readily extended to any peptide with a free carboxyl group. Fast atom bombardment‐collision activated dissociation‐linked field (B/E) scan mass spectrometry unambiguously locates the two 18 O atoms in the carboxyl group of the peptide and not in a peptide amide bond or tyrosine side chain. The two 18 O‐labeled peptide internal standards were used to measure, in a structurally unambiguous fashion, endogenous leucine enkephalin and methionine enkephalin in thalamus tissue at the ppb level. A microcomputer was used for data acquisition and reduction.