Comprehensive profiling and quantitation of amine group containing metabolites.

Primary and secondary amines, including amino acids, biogenic amines, hormones, neurotransmitters, and plant siderophores, are readily derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using easily performed experimental methodology. Complex mixtures of these amine derivatives can be fractionated and quantified using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Upon collision induced dissociation (CID) in a quadrupole collision cell, all derivatized compounds lose the aminoquinoline tag. With the use of untargeted fragmentation scan functions, such as precursor ion scanning, the loss of the aminoquinoline tag (Amq) can be monitored to identify derivatized species; and the use of targeted fragmentation scans, such as multiple reaction monitoring, can be exploited to quantitate amine-containing molecules. Further, with the use of accurate mass, charge state, and retention time, identification of unknown amines is facilitated. The stability of derivatized amines was found to be variable with oxidatively labile derivatives rapidly degrading. With the inclusion of antioxidant and reducing agents, tris(2-carboxyethyl)-phosphine (TCEP) and ascorbic acid, into both extraction solvents and reaction buffers, degradation was significantly decreased, allowing reproducible identification and quantification of amine compounds in large sample sets.

Drought responses of leaf tissues from wheat cultivars of differing drought tolerance at the metabolite level.

Drought has serious effects on the physiology of cereal crops. At the cellular and specifically the metabolite level, many individual compounds are increased to provide osmoprotective functions, prevent the dissociation of enzymes, and to decrease the number of reactive oxygen species present in the cell. We have used a targeted GC-MS approach to identify compounds that differ in three different cultivars of bread wheat characterized by different levels of tolerance to drought under drought stress (Kukri, intolerant; Excalibur and RAC875, tolerant). Levels of amino acids, most notably proline, tryptophan, and the branched chain amino acids leucine, isoleucine, and valine were increased under drought stress in all cultivars. In the two tolerant cultivars, a small decrease in a large number of organic acids was also evident. Excalibur, a cultivar genotypically related to Kukri, showed a pattern of response that was more similar to Kukri under well-watered conditions. Under drought stress, Excalibur and RAC875 had a similar response; however, Excalibur did not have the same magnitude of response as RAC875. Here, the results are discussed in the context of previous work in physiological and proteomic analyses of these cultivars under drought stress.