Fragmentation pathway for glutamine identification: Loss of 73 da from dimethylformamidine glutamine isobutyl ester

Abstract

A fragmentation mechanism for the neutral loss of 73 Da from dimethylformamidine glutamine isobutyl ester is investigated. Understanding this mechanism will allow to improve the identification and quantification of 15N-labeled and unlabeled glutamine and the distinguishing of glutamine and glutamic acid by electrospray ionization (ESI)-tandem mass spectrometry. Before mass spectrometry analysis, glutamine and glutamic acid are derivatized with dimethylformamide dimethyl acetal and isobutanol to form dimethylformamidine isobutyl ester. Derivatization conditions are modified based on an existing method to ensure complete derivatization of glutamic acid and to prevent the hydrolysis of glutamine. The fragmentation mechanism of dimethylformamidine glutamine isobutyl ester is studied and possible fragmentation pathways are proposed. Based on the fragmentation mechanism, a quantification method is developed to quantify both 15N-labeled and unlabeled glutamine and glutamic acid at a series of different neutral losses by performing multiple-reaction monitoring (MRM) scans in a triple-quadrupole mass spectrometer. Labeled glutamine includes 15N-amide labeled, 15N-amine labeled glutamine and glutamine 15N-labeled at both amide and amine positions. Deuterium labeled glutamine and glutamic acid are used as internal standards. Isotope effects are characterized for 15N labeled and deuterium labeled glutamine. It is found that the same method can be used to distinguish aspartic acid from asparagine. This study will improve the application of MS/MS for amino acid quantification and stable isotope labeling metabolism studies.

Discovery of an alternate metabolic pathway for urea synthesis in adult Aedes aegypti mosquitoes

“…For the quantification of labeled amino acids in the feces, 50 l of feces in solution was mixed with 25 l of 1 mM deuterium-labeled amino acids (Cambridge Isotope Laboratories). Then the solution was dried and derivatized as described previously (21,22). For the quantification of unlabeled and labeled urea, 50 l of feces in solution was mixed with 25 l of 1 mM [ 13 C-15 N2]-urea and dried.…”

Analysis of whole body ammonia metabolism in Aedes aegypti using [15N]-labeled compounds and mass spectrometry

“…Then, the solution was dried using a vacuum centrifuge and derivatized as dimethylformamidine isobutyl esters or as isobutyl esters as described previously (Zhang et al, 2005). Briefly, samples mixed with deuterium labeled amino acid standards ([ 2 H 5 ]-Gln, [ 2 H 5 ]-Glu and [ 2 H 4 ]-Ala) were treated with 240 ml of dimethylacetal dimethylformamide (DMF-DMA)-acetonitrile-methanol (2:5:5 by volume) for 10 min at room temperature.…”

“…As a prelude to this work, we have recently investigated the fragmentation mechanism of derivatized glutamine and developed a method to identify and quantify both 15 N-labeled and unlabeled glutamine (labeled glutamine includes 15 N-amide labeled, 15 N-amine labeled and 15 N-labeled at both amide and amine positions) and glutamic acid at a series of different neutral losses by performing multiplereaction monitoring scans in a triple-quadrupole mass spectrometer (Zhang et al, 2005). The results presented in this paper confirm and extend the role of the glutamine synthetase (GS)-glutamate synthase (GltS) reactions as the main metabolic pathway involved in ammonia metabolism in mosquitoes.…”

Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

“…The term ammonia is defined here as NH 3 or NH 4 + or a combination of both. The application of isotopically labeled compounds and mass spectrometry, as well as RNA interference (RNAi), has demonstrated that A. aegypti females efficiently detoxify ammonia through 3 phases: fixation, assimilation, and excretion (5)(6)(7)(8)(9)(10). During this complex metabolic challenge, female mosquitoes synthesize specific amino acids, such as Gln, Glu, Ala, and Pro, through reactions catalyzed by several enzymes, including glutamine synthetase, glutamate synthase, glutamate dehydrogenase, pyrroline-5-carboxylate synthase, pyrroline-5-carboxylaye reductase, and alanine aminotransferase (ALAT).…”