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 ¹⁵N-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 ¹⁵N label distribution. The fragmentation pathways of the nitrogen moieties of glutamine (Gln) and asparagine (Asn) were studied in detail using all mono ¹⁵N isotopomers, which led to a method for differentiating between ¹⁵N-amide and ¹⁵N-amino labeling. The fragmentation involving the amino and amide groups of Gln 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 ¹⁵N abundance and distribution between the amino and amide groups. The detection limit (3 SD) for the presence of a ¹⁵N 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% 15N 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.