Direct analysis of 15N-label in amino and amide groups of glutamine and asparagine

A novel method for on-line determination of the amount and position of N-15-labeling in complex mixtures of amino acids is presented. Underivatized amino acids were analyzed by ion-pair chromatography in combination with mass spectrometry. This enables the direct determination of the N-15 label distribution. The fragmentation pathways of the nitrogen moieties of glutamine (Gin) and asparagine (Asn) were studied in detail using all mono N-15 isotopomers, which led to a method for differentiating between N-15-amide and N-15-amino labeling. The fragmentation involving the amino and amide groups of Gin led to distinct ion structures. The equivalent fragmentation pattern was not observed for Asn. Instead, the amide group of Asn was eliminated as HNCO in a secondary process. The developed analytical method was evaluated by analysis of a range of standard mixtures taking into account different levels of N-15 abundance and distribution between the amino and amide groups. The detection limit (3 SD) for the presence of a N-15 label was 0.7 and 1.0% for Gln and Asn, respectively. The determination of the positional labeling follows a nonlinear function. A representative example at 30% N-15 was used as a benchmark resulting in average relative standard deviations of 2.7 and 15% for Gln and Asn, respectively. The corresponding expectation windows for the positional labeling were found to be 2 and 12%, respectively. Copyright (c) 2006 John Wiley & Sons, Ltd.