~15N/~14N Position-Specific Isotopic Analysis of Polynitrogenous Amino Acids

摘要

15~N/14~N isotope ratios are widely used to study processes and systems involving amino acids. Nitrogen isotope fractionation in biological processes occurs primarily at sites of bond-breaking and formation; the finest discrimination for "isotopic fingerprinting" and studies of isotopic fluxes is thus obtained at the position-specific level. While there are numerous reports of natural intramolecular carbon isotope variability, there are no literature reports of 15~N/14~N position-specific isotopic analysis (N-PSIA) of biologically relevant molecules. We report a methodology for high-precision N-PSIA of four polynitrogenous alpha-amino acids (asparagine, glutamine, lysine, histidine) and the first survey of natural intramolecular 15~N/14~N in these biomolecules. Selective liberation of N-atoms from multiple commercial standards of each parent amino acid was achieved by an appropriate enzymatic reaction or by acid hydrolysis. 15~N/14~N measurements were performed on N-ethoxycarbonyl ethyl ester derivatives of the parent amino acids and their analogues by gas chromatography combustion isotope ratio mass spectrometry, and the average precision for replicate injections was found to be SD(delta(15~N)) = 0.3 per thousand. Position-specific delta(15~N) values of the parent amino acid were directly observed or indirectly calculated using mass balance. The average precision obtained for directly measured positions was SD(delta(15~N)) = 0.2-0.4 per thousand. The average precision for indirectly obtained positions was SD(delta(15~N)) = 0.6-1.3 per thousand as a result of propagation of errors. Enrichment in the side chain-N with respect to the peptide-N was observed in nearly all of the amino acid sources, most notably in asparagine (average DELTA delta_(side-peptide) = + 11 per thousand), which may be indicative of its method of production. In some cases, it was possible to distinguish commercial sources by N-PSIA that could not be distinguished at the compound-specific level.