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Amino‐group‐specific natural abundance nitrogen isotope ratio analysis in amino acids
Author(s) -
Zhang Lin,
Altabet Mark A.
Publication year - 2008
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.3393
Subject(s) - chemistry , isotope ratio mass spectrometry , isotope analysis , amino acid , lysine , isotopes of nitrogen , tryptophan , chromatography , mass spectrometry , nitrogen , organic chemistry , biochemistry , ecology , biology
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 NO 2 −by hypochlorite at high pH. The NO 2 −produced was then quantified colorimetrically with excess hypochlorite quenched using arsenite. Subsequently, buffered azide was used to reduce NO 2 −to N 2 O for isotope ratio analysis using a purge‐and‐trap isotope ratio mass spectrometer. In the case of glycine δ 15 N, the average precision was SD = 0.3‰. Reaction yields and labeling experiments show that this oxidation reaction is highly specific, targeting the α ‐amino group (peptide‐N) of most poly‐N AAs. This permits specific determination of the δ 15 N 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 δ 15 N 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 δ 15 N of amino‐N in complex sample matrices that have not been susceptible to previous approaches. Copyright © 2008 John Wiley & Sons, Ltd.