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Preparation of stable isotope‐incorporated peptide internal standards for field desorption mass spectrometry quantification of peptides in biologic tissue
Author(s) -
Desiderio Dominic M.,
Kai Masaaki
Publication year - 1983
Publication title -
biomedical mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 0306-042X
DOI - 10.1002/bms.1200100806
Subject(s) - chemistry , peptide , mass spectrometry , stable isotope ratio , chromatography , enkephalin , leucine , peptide bond , amide , isotope , biochemistry , amino acid , physics , receptor , quantum mechanics , opioid
Abstract 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.