Amino‐group‐specific natural abundance nitrogen isotope ratio analysis in amino acids

Abstract

Amino acid (AA) nitrogen (N) stable isotope ratio analysis has found a wide variety of important applications including indication of the trophic level of an organism, tracing N transfer within food webs, and monitoring of AA resynthesis during heterotrophic microbial reworking of organic matter. Despite its utility, the current methodology is difficult to employ consistently for natural abundance level precision. Here, we report a new and robust method for high-precision N-compound-specific isotope analysis (N-PCIA) for single-N-containing AAs and N-position-specific isotope analysis (N-PSIA) for poly-N AAs. First the amino-N in AAs was liberated and oxidized to NO2(-) by hypochlorite at high pH. The NO2(-) produced was then quantified colorimetrically with excess hypochlorite quenched using arsenite. Subsequently, buffered azide was used to reduce NO2(-) to N2O for isotope ratio analysis using a purge-and-trap isotope ratio mass spectrometer. In the case of glycine delta15N, the average precision was SD = 0.3 per thousand. Reaction yields and labeling experiments show that this oxidation reaction is highly specific, targeting the alpha-amino group (peptide-N) of most poly-N AAs. This permits specific determination of the delta15N of peptide-N in arginine, tryptophan, and histidine. In the case of lysine, however, the side-chain amino group was found to be partially labile to hypochlorite oxidation. Using isotope fractionation factors estimated from single-N analogues of lysine, the intramolecular delta15N of lysine was calculated by mass balance, and this generally agreed with results for the same sample material analyzed by a previously published enzymatic method. Our method has the advantages of being relatively rapid, robust, and applicable to all poly-N AAs. We have also found it to work well for determining total delta15N of amino-N in complex sample matrices that have not been susceptible to previous approaches.